CN111094986B

CN111094986B - Methods of directing fluid therapy based on adrenomedullin precursors

Info

Publication number: CN111094986B
Application number: CN201880059366.2A
Authority: CN
Inventors: D·威尔逊
Original assignee: BRAHMS GmbH
Current assignee: BRAHMS GmbH
Priority date: 2017-09-13
Filing date: 2018-09-13
Publication date: 2023-11-10
Anticipated expiration: 2038-09-13
Also published as: JP2020537117A; WO2019053116A1; US20200271667A1; WO2019053115A1; EP3682245A1; BR112020004138A2; CN111094986A; CN111065927B; WO2019053116A8; CN111065927A; JP7366007B2; RU2020111719A3; CA3075440A1; JP2023181226A; JP7329503B2; US20200300864A1; RU2020111719A; EP3682246A1; JP2020533604A; JP7544879B2

Abstract

The present invention relates to a method of therapy guidance, stratification and/or monitoring of fluid therapy based on adrenomedullin precursor (proADM) levels. Accordingly, the present invention relates to a method of therapy guidance, stratification and/or monitoring of a fluid therapy comprising providing a sample of said patient, determining the level of an adrenomedullin precursor (proADM) or one or more fragments thereof in said sample, wherein said level of proADM or one or more fragments thereof is indicative of the prescription of a fluid therapy to be administered to said patient. The invention also relates to methods of directing the volume of fluid therapy and methods of treating a disease using fluid therapy based on stratification of proADM in a patient based on the methods described herein.

Description

Methods of directing fluid therapy based on adrenomedullin precursors

Technical Field

The present invention relates to a method of therapy guidance, stratification and/or monitoring of fluid therapy based on adrenomedullin precursor (proADM) levels. Accordingly, the present invention relates to a method for therapy guidance, stratification and/or monitoring of a fluid therapy comprising providing a sample of said patient, determining the level of adrenomedullin precursor (proADM) or one or more fragments thereof in said sample, wherein said level of proADM or one or more fragments thereof is indicative of the prescription of a fluid therapy to be administered to said patient. The invention also relates to a method for guiding the volume of a fluid therapy and a method for treating a disease based on layering proADM on a patient according to the methods described herein using a fluid therapy.

Background

The fluid and electrolyte levels in the body remain relatively constant through several steady state mechanisms. Normally, fluids are obtained from human food and beverage intake, containing small amounts from carbohydrate metabolism. Fluid is lost through urine, sweat and feces and through loss of lung and skin. In healthy individuals, volume homeostasis is regulated primarily by anti-diuretic hormone (ADH). The osmoreceptors and baroreceptors detect a small drop in osmotic pressure and blood pressure, triggering the release of ADH. This causes thirst and reduces the excretion of water by the kidneys.

However, the homeostatic mechanism may not work well after injury (due to trauma or surgery) or during the onset of sepsis or other major illness. Administration of fluid therapy is used to maintain steady state when fluid intake is insufficient, e.g., to supplement any additional fluid loss. These losses may be due to the gastrointestinal tract (vomiting, diarrhea) or urinary tract (diabetes insipidus) or may be caused by blood loss due to trauma or surgery. In addition, as the barrier function of the skin is impaired, the loss of non-sensing may increase during fever or after suffering from burns.

During illness, during surgery, anesthesia, or due to inflammatory conditions (e.g., sepsis), fluid can accumulate into a space (e.g., the peritoneal cavity or pleural cavity) that typically contains a minimum volume of fluid. This is called the third interval and is caused by vasodilation and leakage of the vascular epithelial wall. This disruption to the integrity of the normal compartment can result in a loss of circulatory vessel content. To address these potentially dangerous changes in fluid homeostasis, fluid therapies are often administered to patients with various medical conditions.

Emerging evidence suggests that the type and dosage of fluid resuscitation can affect the prognosis of a patient. If the patient is experiencing fluid (volume) depletion, the patient's heart rate will increase to increase cardiac output and raise blood pressure, thereby maintaining oxygenation of the tissue, but will further stress the patient. Urine becomes concentrated in the event of volume depletion, with more severe cases resulting in decreased urine output. Elevated plasma urea (above 6 mmol/L) and sodium levels (above 145 mmol/L) also create potential adverse events to the patient's health when fluid levels are depleted (journal of pharmacy (The Pharmaceutical Journal), month 9 of 2008, intravenous fluid therapy-background and principle (Intravenous fluid therapy-Background and Principles)).

Some pathological conditions require special consideration. For example, patients with severe burns require relatively large amounts of fluid, calculated from body weight and the percentage of body surface area affected. In traumatic brain injury, the fluid volume may be adjusted according to the mean arterial pressure, which is related to the cerebral perfusion pressure. A relatively large amount of IV fluid is often also required after a wound or sepsis. Fluid administration should be particularly carefully balanced for individuals with heart failure, renal insufficiency, or overt respiratory failure.

Many complications may occur due to fluid therapy (e.g., administration of excessive fluid) (Cotton et al, the cellular, metabolic and systemic consequences of aggressive fluid resuscitation strategies (The cellular, metabolic and systemic consequences of aggressive fluid resuscitation strategies) & Shock & lt & gt 2006, 26:115-21). When this occurs, a number of serious side effects may occur, for example, the heart may not be able to pump an expanded circulatory volume effectively, leading to heart failure and thus pulmonary oedema. Renal failure and pre-existing ventricular damage exacerbate this condition. Abdominal compartment syndrome and acute respiratory distress syndrome are well known consequences of excessive fluid resuscitation and fluid overload. Special care must be taken in treating any patient with heart failure or respiratory failure or at risk of hemodynamic instability. Risks are also associated with too fast correction of disturbances in sodium levels.

Although fluid overload has traditionally been defined as a 10% weight gain in admission, many studies report that fluid overload has a significant correlation with higher mortality and morbidity between ICU patients, regardless of the definition of fluid overload (Frazee et al, critical patient fluid management: a review of the status quo of fluid therapy in intensive care units (Fluid Management for Critically Ill Patients: A Review of the Current State of Fluid Therapy in the Intensive Care Unit), 2016, kidney Diseases (Kidney Diseases), 2 (2), 64-71). Improper fluid prescription does lead to improper management of fluid therapy and the consequences can be fatal. Thus, improving the fluid formulation can avoid preventable complications and bring better health results (Gao et al, journal of clinical pharmacy and therapeutics (Journal of Clinical Pharmacy and Therapeutics), 2015, 40, 489-495).

Currently, there is no objective or reliable way to determine how much fluid should be administered to a patient, nor is there an objective or reliable way to determine which fluid to use. Accordingly, there is an urgent need to develop diagnostic and prognostic tools for assessing fluid therapies and to provide means for therapy guidance, stratification and/or monitoring of fluid therapies that achieve effective and safe fluid levels to be administered.

Disclosure of Invention

In view of the difficulties in the prior art, a potential technical problem of the present invention is to provide means for therapy guidance, stratification and/or monitoring of fluid therapy that achieves an effective and safe level of fluid to be administered.

The solution to the technical problem of the present invention is provided in the independent claims. Preferred embodiments of the invention are provided in the dependent claims.

The present invention thus relates to a method for therapy guidance, stratification and/or monitoring of fluid therapy comprising

-providing a sample of the patient in question,

determining the level of adrenomedullin precursor (proADM) or one or more fragments thereof in said sample,

-wherein the level of proADM or one or more fragments thereof is indicative of a prescription of fluid therapy to be administered to the patient.

It has surprisingly been found that the level of proADM determined in a sample obtained from a patient can be used to monitor and guide the fluid therapy being administered. Excessive administration of fluid volumes is detrimental, resulting in organ dysfunction and progression to multiple organ failure and eventual death. Thus, it is important to administer the correct volume of liquid on a patient basis. As shown in more detail in the data below, the proADM level in a patient sample correlates with the amount of fluid administered to the patient over time and additionally provides prognostic information regarding the likelihood of adverse events occurring in the patient's health. Based on the data presented herein, proADM levels are positively correlated with the amount of fluid administered, such that high proADM levels (severe levels) are evident in patients treated with relatively large volumes of liquid. By monitoring proADM levels, adjustments can be made to the type and amount of fluid administered, potentially reducing the risk of fluid overload in patients receiving fluid therapy.

In some embodiments, the invention describes using MR-proADM concentrations from samples at, for example, 1, 2, or 3 time points (e.g., at baseline, day 1, and day 4) to direct the volume of fluid to be administered to maintain low MR-proADM concentrations or to reduce MR-proADM concentrations and to direct the use of specific colloids according to MR-proADM concentrations. By using such a method, the risk of adverse events (e.g., those adverse events related to fluid overload, or other adverse events related to any given medical condition to which the patient is suffering) due to fluid therapy adjustments can be managed and even reduced based on the indication provided by the proADM level.

As shown in more detail below, while proADM shows some advantages, MR-proADM and lactate similarly function in guiding the volume of fluid to be administered during the first 24 hours after receiving fluid therapy. MR-proADM is significantly more accurate in guiding the volume of fluid to be administered between 24 and 96 hours. Quite unexpectedly, proADM will represent a fluid therapy improvement means for monitoring a given marker (e.g., lactate).

As used herein, the term fluid therapy relates to the administration of a liquid to a subject in need thereof. The term fluid replenishment or fluid resuscitation may also be used interchangeably. Under fluid therapy, various treatments are envisaged, such as supplementing the fluid with oral rehydration therapy (drinking water), intravenous therapy, rectally, or injecting the fluid into subcutaneous tissue. Intravenous methods are preferred.

The fluid to be administered encompasses any liquid that facilitates maintenance or attainment of a healthy fluid level and/or balance (steady state) in the subject. Preferably, established colloidal fluid therapy or crystal fluid therapy is administered.

The term "prescription of fluid therapy" includes any indication, information, and/or description regarding the fluid therapy to be administered, including but not limited to the volume, dosage, rate, mode of administration, and/or type of therapeutic fluid to be administered.

In one embodiment of the method, the patient has been diagnosed as having a medical condition requiring fluid therapy, such as wherein the patient has been diagnosed as critical.

In embodiments of the present invention, in any medical setting, patients exhibiting symptoms or absence of any physiological condition may be examined. According to a preferred embodiment, the sample is separated from the patient during the medical examination.

In embodiments of the invention, the patient is or has been diagnosed with a critical condition. According to further embodiments, the sample is isolated from the patient at or after the diagnostic time point. Furthermore, in an embodiment of the method of the invention, the medical treatment has already been started at or before the diagnostic time point. In an embodiment, the patient has been diagnosed as critical and the medical treatment has been initiated. The sample may be isolated from the patient before, at or after the start of the diagnosis and treatment.

In one embodiment of the method, the patient has been diagnosed as having an infectious disease, one or more organ failure, and/or wherein the patient is a post-traumatic or post-operative patient.

Organ failure may occur, for example, during sepsis or during other diseases in which the patient is critically ill and may be associated with failure of the kidney, liver and/or blood coagulation system, but is not limited thereto. Patients experiencing organ failure typically require fluid therapy, although to date, only a specific prescription of the therapy has been estimated for any given patient. Post-traumatic patients (e.g., patients suffering from severe injuries (e.g., multiple trauma)) or post-operative patients may often have low body fluid content due to blood loss and also require fluid resuscitation.

In one embodiment of the method, the patient has been diagnosed with sepsis, severe sepsis, or septic shock. Sepsis patients typically receive fluid therapy.

In general, there has been no significant change in the treatment of sepsis over the last few decades, and therapies currently in use include antibiotics, source control, fluid resuscitation, and vasopressors. Fluid resuscitation has long been a permanent means of treating sepsis, even earlier than antibiotics. Despite the widespread use of fluid resuscitation, there is still a contradiction in clinical evidence supporting fluid resuscitation in sepsis due to lack of well-controlled studies and uncertainties regarding the type and volume of liquid to be administered. The present invention provides an unexpected and highly beneficial method of assessing the correctness or appropriateness of an administered fluid and enabling adjustment of a treatment based on proADM levels. Directing fluid administration in such ways and the like represents an entirely new approach for effectively managing fluid therapy.

In one embodiment, the prescription of fluid therapy includes an indication of the type and/or dosage of fluid therapy, or wherein the patient receives fluid therapy and the prescription of fluid therapy is whether a change in fluid therapy is required. Thus, a change in fluid therapy may involve any change in volume, dose, rate, mode and/or type of fluid to be administered.

In one embodiment, the prescription of the fluid therapy includes a fluid volume, frequency, and/or rate indicative of a fluid to be administered to the patient.

As shown in more detail below, the volume of fluid administered to the patient within about 24 hours or within about 4 days correlates with the level of proADM measured in the sample tested. As such, a high volume of fluid that has been administered is associated with an elevated proADM level that is associated with an increased risk of adverse events to the patient. The invention thus achieves that the time required for the treatment of the first and second objects can be reduced over a period of time (e.g., within 1 to 24 hours) or over other periods of time (e.g., within 1 to 7 days, preferably within 3 to 5 days, or about 4 days) or within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 60 hours, or 59 hours. The skilled person can then adjust the amount of volume applied to achieve the appropriate level in a time period such as those described above.

In one embodiment, the method indicates that the fluid therapy includes administration of a colloidal solution. Colloids are dispersions of large organic molecules (e.g., gelofusin, voluven). Colloids are generally suspensions of molecules in a carrier solution that, due to their molecular weight, are relatively unable to cross healthy semipermeable capillary membranes.

In one embodiment, the method indicates that the fluid therapy comprises a colloid or blood selected from gelatin, albumin and/or starch solutions or a blood-derived fluid.

In one embodiment, the method indicates that the fluid therapy includes administering a crystalline solution. Crystals are aqueous solutions of small molecules (e.g., sodium chloride, glucose, hartmann's solution). The crystals are typically solutions that are freely permeable but contain ions of sodium and chloride at concentrations that determine the tonicity of the fluid.

In one embodiment, the method further comprises determining lactate levels in a sample isolated from the patient. Lactate is a biomarker that is typically elevated in sepsis, for example, although the reasons are not clear. Although there are many reasons for the possible increase in lactate levels in sepsis patients, in the early manifestations of these patients, oxygen deficiency is the most likely cause. Although there is no exact guideline for administering fluid therapy based on lactate levels, fluid resuscitation has been shown to be associated with improved efficacy in sepsis patients with moderate lactate values. The present invention provides complementary biomarkers (proADM) that provide similar but improved fluid therapy guidance independent of lactate levels. However, lactate levels do also achieve guidelines for the volume of fluid to be administered within the first 24 hours after receiving fluid therapy, and as such, combined assessment of lactate levels and proADM levels is a preferred embodiment of the present invention.

As described in more detail below, the present invention proposes a defined severity level of proADM levels and subsequent guidance for managing fluid therapies based on these levels.

In one embodiment of the method, the patient is receiving fluid therapy and a medium or high severity level of proADM or one or more fragments thereof determined in the sample is indicative of an adverse event,

wherein the moderate severity level of proADM or one or more fragments thereof is 2.75 nmol/l.+ -. 20% to 10.9 nmol/l.+ -. 20%,

and wherein the high severity level of proADM or one or more fragments thereof is higher than 10.9nmol/l ± 20%.

According to the invention, the adverse event is preferably death, 28-day death (possibly within 28 days after proADM evaluation), organ failure, preferably renal failure. Thus, the present invention provides an assessment of fluid therapy and indicates changes if necessary and additionally provides an assessment of the likelihood of adverse events (e.g., death within 28 days) occurring. Based on the assessment caused by proADM levels, fluid therapy can then be adjusted to improve (preferably reduce) proADM levels and thereby reduce the risk of subsequent adverse events.

In one embodiment, the patient receives fluid therapy and a medium or high severity level of proADM or one or more fragments thereof determined in the sample is indicative of a decrease in the volume and/or rate of fluid to be administered to the patient,

A common and unfortunate situation in treating patients (e.g., sepsis) is that the volume or rate of fluid administered is too high. If the patient exhibits a proADM level at a high or moderate severity level, this provides an indication to the physician to reduce the amount or frequency of fluid to be administered to correct and hopefully reduce the proADM level, thereby improving prognosis.

Based on the data presented herein, proADM levels are positively correlated with the amount of fluid administered, such that high proADM levels (severe levels) are evident in patients treated with relatively large volumes of liquid. A high proADM level (high severity level) thus also indicates an increased risk of future adverse events.

Thus, the present method involves using proADM as a marker of fluid overload.

Thus, the present method involves the use of proADM as a marker for predicting the risk of adverse events in patient health related to fluid overload.

In some embodiments, it is particularly important to detect changes or increases in proADM levels over time in order to take appropriate measures in adjusting the treatment.

In one embodiment, the method comprises

-determining the level of proADM or one or more fragments thereof in a first sample and a second sample from the patient, wherein the second sample is obtained after obtaining the first sample;

-wherein no change or an increase in the level of proADM or one or more fragments thereof in the second sample compared to the first sample is indicative of a decrease in the volume, frequency and/or rate of the fluid to be administered to the patient.

In some embodiments, the first sample is isolated at or before the beginning of fluid therapy (time point 0) and the second sample is isolated at least 30 minutes, preferably 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 7 days, or 10 days after the beginning of the therapy.

In some embodiments, the first sample is isolated at or before the beginning of fluid therapy (time point 0) and the second sample is isolated at a time point between 12 hours and 36 hours, preferably 24 hours after the beginning of the therapy and/or at a time point between 3 days to 5 days, preferably 4 days after the beginning of the therapy.

In one embodiment, an increase in proADM or one or more fragments thereof from a low severity level to a medium or high severity level or an increase in proADM or one or more fragments thereof from a medium severity level to a high severity level indicates a decrease in the volume and/or rate of the fluid to be administered to the patient, wherein

A low severity level of proADM or one or more fragments thereof of less than 2.75 nmol/l.+ -. 20%,

a moderate severity level of 2.75 nmol.+ -. 20% to 10.9 nmol.+ -. 20% of proADM or one or more fragments thereof, and

the high severity level of proADM or one or more fragments thereof is higher than 10.9 nmol/l.+ -. 20%.

In the embodiments described herein, the severity level is preferably defined by a threshold value representing a boundary between a low severity level, a medium severity level, or a high severity level. Thus, any embodiment that presents a threshold may use a single threshold format as a boundary between two severity levels or a single threshold level for each severity level.

In some embodiments, the proADM threshold between low and medium severity levels is:

2.75 nmol/l.+ -. 20% or 2.75 nmol/l.+ -. 15% or.+ -. 12%,.+ -. 10%,.+ -. 8% or.+ -. 5%,

And the proADM threshold between medium and high severity levels is:

10.9 nmol/l.+ -. 20% or 10.9 nmol/l.+ -. 15% or.+ -. 12%,.+ -. 10%,.+ -. 8% or.+ -. 5%.

These thresholds are preferably related to the assessment of proADM severity level at baseline (in other words, after diagnosis and/or initiation of therapy and/or hospitalization). The baseline level itself can indicate the fluid therapy prescription by, for example, a single measurement or multiple measurements taken at an initial single point in time.

2.80 nmol/l.+ -. 20% or 2.80 nmol/l.+ -. 15% or.+ -. 12%,.+ -. 10%,.+ -. 8% or.+ -. 5%,

and the proADM threshold between medium and high severity levels is:

9.5 nmol/l.+ -. 20% or 9.5 nmol/l.+ -. 15% or.+ -. 12%,.+ -. 10%,.+ -. 8% or.+ -. 5%.

These thresholds are preferably related to the assessment of proADM severity level after 1 day (in other words, about 24 hours after baseline, in other words, about 1 day after diagnosis and/or treatment initiation and/or hospitalization). For example, in embodiments where proADM is measured one day after initiation of treatment, the threshold on day 1 may be employed. As is apparent from the above, the critical value between the medium and high levels is slightly lower than at baseline, i.e. even lower (but still relatively higher) levels over time are also associated with high risk and are classified as high severity levels.

and the proADM threshold between medium and high severity levels is:

7.7 nmol/l.+ -. 20% or 7.7 nmol/l.+ -. 15% or.+ -. 12%,.+ -. 10%,.+ -. 8% or.+ -. 5%.

These thresholds are preferably related to the assessment of proADM severity level after 4 days (in other words, about 4 days after baseline, in other words, about 4 days after diagnosis and/or treatment initiation and/or hospitalization). For example, in an embodiment where proADM is measured 4 days after initiation of treatment, the threshold on day 4 may be employed. As is apparent from the above, the threshold between medium and high levels is slightly lower than at baseline or day 1, i.e. even lower (but still relatively higher) levels over time are also associated with high risk and are classified as high severity levels.

In some embodiments, the threshold level employed in the above embodiments may be adjusted according to an appropriate level depending on the date on which the measurement is made. Each of the thresholds is subject to some variation due to common variances as may be desired by the skilled person. As presented below, the relevant critical level is determined based on a large amount of data, but is not intended to be the final or exact value in all possible embodiments. Similar results can be expected by using similar threshold values to those listed, i.e., within ±20%, ±15%, ±12%, ±10%, ±8% or ±5%, as can be determined by the skilled person.

Any embodiment listing ±20% of a given threshold may be considered as also disclosing ± 15%, ±12%, ±10%, ±8% or ± 5%.

Any embodiment listing a particular threshold on day 1 or day 4 may be considered to also disclose the corresponding threshold on other days, e.g., an embodiment listing a baseline threshold may be considered to also refer to the same embodiment listing a day 1 or day 4 threshold.

The threshold value is in a preferred embodiment applicable to a blood sample or a blood-derived sample, but is not limited thereto.

Further aspects of the invention relate to methods for indicating therapy guidelines for fluid therapy based on proADM levels. Specifically, in some embodiments, a prescription may be made for the volume of fluid to be administered based on the measured proADM value.

In one embodiment of the method, the patient receives fluid therapy, and

a low severity level of proADM or one or more fragments thereof determined in the sample indicates that 2.78ml/kg + 20% or less of fluid (fluid administered per kilogram of body weight of the patient) is administered to the patient within about 24 hours,

-a medium severity level of proADM or one or more fragments thereof determined in the sample indicates that 4.94ml/kg ± 20% or less of a fluid, preferably 4.2ml/kg ± 20% of a fluid, or

A high severity level of proADM or one or more fragments thereof determined in the sample indicates that less than 9.95ml/kg ± 20% of the fluid, preferably 7.15ml/kg ± 20% of the fluid is administered to the patient within about 24 hours,

and is also provided with

Wherein the low severity level of proADM or one or more fragments thereof is below 2.75nmol/l + -20%,

The above values relate to a guideline for therapy based on the volume of fluid administered within about 24 hours from baseline.

To the inventors' knowledge, therapy guideline embodiments, such as these therapy guideline embodiments, represent the first objective biomarker-based fluid therapy volume guideline in the field of fluid therapy management.

In some embodiments, the fluid to be applied comprises or consists of a colloid and optionally crystals. The values provided herein are preferably colloid-related, and not crystal-applied.

The indicated fluid volumes or rates to be administered are derived from the data presented below, wherein the average colloid volume administered to the patient is measured and the average value is determined, and these values are related to low, medium and high severity levels of proADM. Thus, for the example in which an increase in proADM level is observed (i.e., an increase in proADM level from a moderate level to a high level), an average amount of fluid administered over 1 day or 4 days may be used as an indicator of an excessive amount of fluid. Thus, guidelines are to administer amounts lower than the average value associated with an increase in proADM.

In one embodiment of the method, the patient receives fluid therapy, and

a low severity level of proADM or one or more fragments thereof determined in said sample indicates that 6.97ml/kg ± 20% or less of a fluid (fluid administered per kilogram of body weight of said patient) is administered to said patient within about 4 days

-a medium severity level of proADM or one or more fragments thereof determined in the sample indicates that 14.47ml/kg ± 20% or less of the fluid, preferably 11.45ml/kg ± 20% of the fluid, or

A high severity level of proADM or one or more fragments thereof determined in the sample indicates that less than 32.30ml/kg ± 20% of fluid, preferably 14.10ml/kg ± 20% of fluid is administered to the patient within about 4 days,

and is also provided with

Wherein the low severity level of proADM or one or more fragments thereof is below 2.8nmol/l + -20%,

wherein the moderate severity level of proADM or one or more fragments thereof is 2.8 nmol/l.+ -. 20% to 9.5 nmol/l.+ -. 20%,

and wherein the high severity level of proADM or one or more fragments thereof is higher than 9.5nmol/l ± 20%.

The above values relate to a guideline for therapy based on the volume of fluid administered within about 4 days from baseline.

Any embodiment listing ±20% of the given fluid application volume can be considered as also disclosing ± 15%, ±12%, 10%, ±8% or ± 5%.

In some embodiments, the crystals may be applied instead of or in parallel with the colloid. If crystals are to be administered, it is generally necessary to administer a volume that is greater than the volumes listed above. In some embodiments, the volumes listed above should be tripled when only crystals are applied instead of colloids. In some embodiments, when only crystals are applied instead of colloids, the volumes listed above should be multiplied by 1.4, 1.5 or 1.6. Some studies showed that 1.4L saline corresponds to 1L albumin. Other studies estimated that 3L crystals corresponded to 1L colloid.

In one embodiment, the method is characterized in that the fluid therapy to be administered to the patient is a starch solution, preferably hydroxyethyl starch.

In one embodiment, the method is characterized in that the fluid therapy to be administered to the patient is an albumin solution, preferably a 20% albumin solution.

In one embodiment, the method is characterized in that the fluid therapy to be administered to the patient is a gelatin solution.

A further aspect of the invention relates to the medical use of a fluid therapy or therapeutic fluid for treating a patient identified using the methods described herein. Comprising a method of treating a medical indication described herein, the method comprising performing the method as described herein and subsequently treating the patient. For example, the treatment may reflect an indication of treatment provided in accordance with the methods of the present invention. The definition of this patient group is novel compared to previous methods. There is no indication in the relevant art that patients with these specific proADM levels will be treatable using the guidelines presented herein.

Accordingly, the present invention relates to a pharmaceutical composition comprising a fluid therapy (therapeutic fluid), such as a colloidal solution and/or a crystalline solution, for use as a medicament in fluid therapy of a patient in need thereof, wherein the composition is administered to the patient after being prescribed for said administration by a method as described herein.

In one embodiment, a pharmaceutical composition for use as an agent in fluid therapy as described herein is characterized by administration of the composition according to stratification of the patient according to the level of proADM or one or more fragments thereof obtained by the method described herein.

In one embodiment, a pharmaceutical composition for use as a medicament in fluid therapy as described herein is characterized by

-the patient has received fluid therapy, and

when the patient exhibits a medium or high severity level of proADM or one or more fragments thereof determined in the sample, the patient receives a decrease in the volume, frequency and/or rate of the fluid administered to the patient,

and wherein the high severity level is higher than 10.9 nmol/l.+ -. 20%.

-the level of proADM or one or more fragments thereof in a first sample and a second sample from said patient has been determined, wherein said second sample is obtained after obtaining said first sample;

-wherein when it is determined that the level of proADM or one or more fragments thereof in the second sample is unchanged or increased compared to the first sample, the patient is administered a reduced volume, frequency and/or rate of the fluid.

Administering 2.78 + -20% or less of the fluid (administered per kilogram of body weight of the patient) to a patient in the sample for which proADM or one or more fragments thereof are determined to have a low severity level within about 24 hours,

administering 4.94ml/kg + -20% or less of fluid, preferably 4.2ml/kg + -20% of fluid, to a patient in the sample for about 24 hours for whom proADM or one or more fragments thereof are determined to have a moderate severity level,

administering less than 9.95ml/kg + -20% of the fluid, preferably 7.15ml/kg + -20% of the fluid, to a patient in the sample for about 24 hours for whom the proADM or one or more fragments thereof are determined to have a high severity level,

And is also provided with

The therapeutic guidelines as described in detail above with respect to the method, with respect to a number of potential thresholds or volumes of treatment suggested depending on date and/or differences are considered to be disclosed and equally applicable to embodiments with respect to the medical uses described herein.

In one embodiment, a pharmaceutical composition for use as a medicament in fluid therapy as described herein is characterized in that the administered fluid comprises or consists of a colloid and optionally a crystal.

In one embodiment, the pharmaceutical composition as described herein for use as a medicament in fluid therapy is characterized in that a starch solution, preferably hydroxyethyl starch, is administered to the patient when determining the low and/or medium severity level of proADM or one or more fragments thereof determined in the sample.

In one embodiment, the method for therapy guidance, stratification and/or monitoring of fluid therapy based on adrenomedullin precursor (proADM) levels is characterized in that the fluid therapy to be administered to the patient is a starch solution, preferably hydroxyethyl starch.

Hydroxyethyl starch (HES/HAES, molar mass 130-200kg/mol (typical value)) sold under the trade name Voluven et al (Hespan, volulyte, tetrahes, hestar) is a nonionic starch derivative for use as a volume expander in fluid therapy, preferably intravenous therapy. Intravenous solutions of hydroxyethyl starch are commonly used to prevent shock after severe blood loss due to trauma, surgery or other problems.

A problem with the past administration of HES is that HES can lead to a need to increase the frequency of renal replacement therapies. High molecular weight HES has been associated with coagulopathy, itching, renal toxicity, acute renal failure, and death. This finding has been apparent from other studies and resulted in a retention when HES is administered to patients in need of fluid therapy or resuscitation (e.g., sepsis patients). The data presented below indicate that this is not necessarily the case, especially when the MR-proADM level is low or at a medium level.

Patients receiving HES with low or moderate proADM levels at baseline did not exhibit unexpectedly high levels of RRT requirements. When the MR-proADM concentration is already high at baseline, RRT is required for all patients to whom HES is administered. Thus, HES can be administered to patients with lower or moderate proADM levels at baseline (preferably, in relatively low amounts) without undue risk of inducing a medical condition requiring RRT.

Thus, the present invention relates to a method for patient stratification based on proADM levels, in particular for identifying patients prone to need RRT or those patients for whom RRT is less desirable, preferably stratification of HES administration. Accordingly, the present invention relates to methods of therapy guidance, stratification and/or monitoring of HES therapies based on proADM levels. This patient stratification enables therapeutic decisions as to whether HES should be administered to the patient and what amounts are. The average fluid volume administered to the patient is indicative of the HES administration prescription to be performed according to the following data.

In one embodiment of the method, the patient receives fluid therapy, and

-a low severity level of proADM or one or more fragments thereof determined in the sample indicates that 3703.75ml/kg ± 20% HES or less, preferably 1713ml/kg ± 20% HES, or

A moderate severity level of proADM or one or more fragments thereof determined in said sample indicates that 3020.27ml/kg ± 20% HES or less, preferably 2224.45ml/kg ± 20% HES, or

And is also provided with

-wherein the low severity level of proADM or one or more fragments thereof is below 2.75nmol/l ± 20%, and

-wherein the moderate severity level of proADM or one or more fragments thereof is 2.75nmol/l ± 20% to 10.9nmol/l ± 20%.

These embodiments are related to unexpectedly low RRT requirements and such that administration of HES does not enter high risk levels for induction of RRT. Any embodiment listing ±20% of a given threshold may be considered as also disclosing ± 15%, ±12%, ±10%, ±8% or ± 5%. Any embodiment listing ±20% of the given fluid application volume can be considered as also disclosing ± 15%, ±12%, 10%, ±8% or ± 5%. The above examples relate to the threshold value of proADM at baseline and the subsequent guidance for the next 3-5 days, preferably about 4 days. As noted above, any embodiment in which a particular threshold value for a baseline is recited may also or alternatively be considered to refer to the same embodiment in which a threshold value for day 1 or day 4 is recited.

In one embodiment, the pharmaceutical composition as described herein for use as a medicament in fluid therapy is characterized in that when determining a high severity level of proADM or one or more fragments thereof determined in the sample, an albumin solution, preferably a 20% albumin solution, is administered to the patient.

In one embodiment, the method for therapy guidance, stratification and/or monitoring of fluid therapy based on adrenomedullin precursor (proADM) levels is characterized in that the fluid therapy to be administered to the patient is an albumin solution, preferably a 20% albumin solution.

With or without shock, 20% of albumin (human) is recommended for emergency treatment of hypovolemia. The effectiveness of the reverse hypovolemia is largely dependent on its ability to circulate interstitial fluid. It is most effective for patients with abundant moisture. When hypovolemia is caused by bleeding, compatible red blood cells or whole blood should be administered in combination as soon as possible. Albumin may be administered to patients with hypovolemia, hypoalbuminemia, ovarian hyperstimulation syndrome, adult Respiratory Distress Syndrome (ARDS), acute kidney disease, diuretic induction or neonatal hemolysis. Albumin formulations containing 15-30% albumin can also be obtained.

Albumin solutions have been used for decades to treat critically ill patients. However, increased mortality has been reported in patients receiving albumin solutions, and the role of albumin administration in critically ill patients has been controversial. Albumin is known to have a variety of physiological effects, including regulation of Colloid Osmotic Pressure (COP), binding and transport of various substances (e.g., drugs, hormones) in the blood, antioxidant properties, nitric oxide modulation and buffering capacity, which are particularly relevant in critically ill patients. It was also determined that the low serum albumin levels common in critically ill patients are associated with poor efficacy. Thus, there is a good theoretical basis for the use of albumin infusion in critically ill patients. However, albumin solutions also have limitations, including high costs relative to possible alternatives (especially crystals) and potential risks of transmission of microorganisms, anticoagulants and allergies. Additional guidelines are required to direct albumin fluid therapy.

The results shown in the data below demonstrate that 20% albumin appears to be a suitable choice for reducing mortality in patients with high ADM concentrations, but it also encourages patients to reach RRT requirements, as is evident from the high RRT frequency required in patients receiving relatively large amounts of albumin. In patients with moderate ADM levels administered with relatively small amounts of albumin, mortality and RRT requirements are very low.

Thus, the present invention relates to a method for stratification of patients based on proADM levels, in particular for identifying patients susceptible to RRT or those desiring lower RRT, preferably stratification of albumin administration. Accordingly, the present invention relates to methods of therapy guidance, stratification and/or monitoring of albumin therapies based on proADM levels. This patient stratification enables a therapeutic decision as to whether and by what amount albumin should be administered to the patient. The average fluid volume administered to the patient is indicative of the albumin administration regimen to be performed according to the following data.

In one embodiment of the method, the patient receives fluid therapy, and

-a low severity level of proADM or one or more fragments thereof determined in said sample indicates that 743.75ml/kg±20% albumin or less, preferably 689.46ml/kg±20% albumin, or

A moderate severity level of proADM or one or more fragments thereof determined in said sample indicates that 1153.95ml/kg±20% albumin or less, preferably 696.96ml/kg±20% albumin, or

A high severity level of proADM or one or more fragments thereof determined in the sample indicates that 3173.86ml/kg ± 20% albumin or less, preferably 1000.83ml/kg ± 20% albumin is administered to the patient within about 4 days (e.g. between 3 days and 5 days),

and is also provided with

These examples are associated with unexpectedly low mortality frequency and low RRT requirements and such that administration of albumin does not enter high risk levels for induction of RRT. Any embodiment listing ±20% of a given threshold may be considered as also disclosing ± 15%, ±12%, ±10%, ±8% or ± 5%. Any embodiment listing ±20% of the given fluid application volume can be considered as also disclosing ± 15%, ±12%, 10%, ±8% or ± 5%. The above examples relate to the threshold value of proADM at baseline and the subsequent guidance for the next 3-5 days, preferably about 4 days. As noted above, any embodiment in which a particular threshold value for a baseline is recited may also or alternatively be considered to refer to the same embodiment in which a threshold value for day 1 or day 4 is recited.

In one embodiment, a pharmaceutical composition for use as an agent in fluid therapy as described herein is characterized in that a gelatin solution is administered to the patient when determining the medium and/or high severity level of proADM or one or more fragments thereof determined in the sample.

In one embodiment, the method for therapy guidance, stratification and/or monitoring of fluid therapy based on adrenomedullin precursor (proADM) levels is characterized in that the fluid therapy to be administered to the patient is a gelatin solution.

Gelatin is often administered as a plasma volume replacement. This means that it supplements the fluid lost from the circulation. A common and commercially available liquid gelatin is gelafusine (gelafusine), which is a 4% w/v succinylated gelatin (also known as modified fluid gelatin) solution used as an intravenous colloid that behaves much like blood filled with albumin. The product is also available under the trade name Isoplex, which also contains an electrolyte. Isoplex can be used for the initial management of hypovolemic shock caused by, for example, bleeding, acute trauma or surgery, burns, sepsis, peritonitis, pancreatitis or crush injury.

Gelatin causes increases in blood volume, blood flow, cardiac output and oxygen transport. Gelatin is used to supplement blood and body fluids lost due to, for example, handling, accidents or burns. Gelatin may be used instead of or as with blood transfusion. Gelatin may also be used to fill circulating blood volume during use of a heart-lung machine or artificial kidney. Gelatin is preferably administered intravenously, i.e. by instillation.

Gelatin can have side effects. In rare cases, mild skin reactions (measles, urticaria) or anaphylactic shock occur. Hypervolemia and excessive water were also observed. It is necessary to monitor therapy, in particular serum ionization diagrams and fluid balance. This is especially true in cases of hypernatremia, dehydration status and renal insufficiency. In the case of coagulation dysfunction and chronic liver disease, the coagulation parameters and serum albumin should be monitored. Since allergic (anaphylactic)/anaphylactoid (anaplactic) reactions may occur, proper monitoring of the patient is necessary. Thus, additional guidelines are also needed to monitor and/or stratify gelatin fluid therapy patients.

The results shown in the data below indicate that gelatin does not appear to be a good choice for patients with low ADM concentrations. Patients receiving gelatin as a fluid therapy, even with lower levels of proADM, have a higher frequency of mortality than one would expect. Mortality in patients with moderate proADM levels who received higher amounts of gelatin was also higher. However, administration of relatively small amounts of gelatin to patients with high proADM levels does result in reduced ADM levels and associated low mortality.

Thus, the present invention relates to a method for stratification of patients based on proADM levels, in particular for identifying patients susceptible to RRT or those desiring lower RRT, preferably stratification of gelatin administration. Thus, the present invention relates to methods of therapy guidance, stratification and/or monitoring of gelatin therapies based on proADM levels. This patient stratification enables a therapeutic decision as to whether and by what amount gelatin should be administered to the patient. The average fluid volume administered to the patient is indicative of the gelatin administration regimen to be performed according to the following data.

In one embodiment of the method, the patient receives fluid therapy, and

-a low severity level of proADM or one or more fragments thereof determined in said sample indicates that 2500ml/kg ± 20% gelatin or less, preferably 1256.46ml/kg ± 20% gelatin, or alternatively, is administered to said patient within about 4 days (e.g. between 3 days and 5 days)

A moderate severity level of proADM or one or more fragments thereof determined in said sample indicates that 2711.33ml±20% albumin or less, preferably 1452ml±20% gelatin, or

A high severity level of proADM or one or more fragments thereof determined in the sample indicates that 4483.93ml±20% gelatin or less, preferably 1163.33ml±20% gelatin, is administered to the patient within about 4 days (e.g. between 3 days and 5 days),

and is also provided with

These examples are associated with unexpectedly low mortality frequency and low RRT requirements and such that administration of gelatin does not enter high risk levels for induction of RRT. Any embodiment listing ±20% of a given threshold may be considered as also disclosing ± 15%, ±12%, ±10%, ±8% or ± 5%. Any embodiment listing ±20% of the given fluid application volume can be considered as also disclosing ± 15%, ±12%, 10%, ±8% or ± 5%. The above examples relate to the threshold value of proADM at baseline and the subsequent guidance for the next 3-5 days, preferably about 4 days. As noted above, any embodiment in which a particular threshold value for a baseline is recited may also or alternatively be considered to refer to the same embodiment in which a threshold value for day 1 or day 4 is recited.

In one embodiment, a pharmaceutical composition for use as a medicament in fluid therapy as described herein is characterized in that the patient receives intravenous administration of the composition.

Intravenous (IV) therapy is a therapy that delivers liquid substances directly into the vein and is the preferred method of administration of the present invention. Intravenous routes of administration may be for injection (with a syringe at higher pressure) or infusion (typically using only pressure provided by gravity). Intravenous infusion is commonly referred to as instillation. Intravenous route is a rapid method of delivering drugs and fluid supplements throughout the body, as the circulation carries these drugs. Intravenous therapy may be used for fluid replenishment (e.g., correction of dehydration) to correct electrolyte imbalance, delivery of drugs, and for blood transfusion or for treatment of other medical conditions described herein.

In one embodiment, the pharmaceutical composition for use as a medicament in fluid therapy as described herein is characterized in that the administered fluid comprises an additional active agent, preferably an antibiotic. This embodiment is particularly preferred in the case of infections such as sepsis or septic shock.

A further aspect of the invention relates to a kit for therapy guidance, stratification and/or monitoring of a fluid therapy comprising:

A detection reagent for determining the level of proADM or one or more fragments thereof in a sample from a subject,

-reference data, such as a reference level, corresponding to a high, medium and/or low severity level of proADM, wherein the low severity level of proADM or one or more fragments thereof is below 2.75nmol/l±20%, wherein the medium severity level of proADM or one or more fragments thereof is between 2.75nmol/l±20% and 10.9nmol/l±20%, and wherein the high severity level of proADM or one or more fragments thereof is above 10.9nmol/l±20%, wherein the reference data is stored on a computer readable medium and/or used in the form of computer executable code configured to compare the determined levels of proADM or one or more fragments thereof, and

preferably a pharmaceutical composition comprising a therapeutic fluid, more preferably a colloidal solution and optionally a crystalline solution.

In addition to the above-described methods of therapy guidance and/or methods of treatment, the present invention further relates to methods of providing an indication of other therapies or initiating additional therapies. For example, in one embodiment, renal replacement therapy is the preferred follow-up therapy in cases where high levels of proADM (moderate or severe levels) are identified or when proADM levels are not reduced over time.

In further embodiments, the treatment that the patient receives includes one or more of the following: antibiotic treatment, invasive mechanical ventilation, non-invasive mechanical ventilation, renal replacement therapy, use of vasopressors, fluid therapy, corticosteroids, blood transfusion or platelet infusion, splenectomy, direct thrombin inhibitors such as lepirudin (lepirudin) or argatroban, blood diluents such as bivalirudin (bivalirudin) and fondaparinux (fondaparinux), heparin, lithium carbonate, folic acid, extracorporeal blood purification and/or organ protection are deactivated in the case of heparin-induced thrombocytopenia.

Preferably, in some embodiments, the methods described herein are performed by determining the level of proADM or one or more fragments thereof, wherein the determining proADM comprises determining the level of MR-proADM in the sample.

The use of a determined MR-proADM is preferred for any given embodiment described herein and thus can be considered in the context of each embodiment. In a preferred embodiment, an "ADM fragment" may be considered as an MR-proADM. In some embodiments, any fragment or precursor of ADM (e.g., pre-pro-ADM), a peptide pro-ADM known per se as ADM, or a fragment thereof (e.g., MR-pro ADM) may be employed.

According to another embodiment of the invention, determining the level of proADM or one or more fragments thereof comprises determining the level of MR-proADM in the sample.

According to the present invention, in the context of "indicating a subsequent adverse event" and "indicating the absence of a subsequent adverse event", the term "indicating" is intended as a measure of risk and/or likelihood. Preferably, the "indication" of the presence or absence of an adverse event is intended as a risk assessment and should not generally be construed in a limiting manner as definitively indicating the absolute presence or absence of the event.

Thus, the term "indicating the absence of a subsequent adverse event" or "indicating a subsequent adverse event" may be understood as indicating a low or high risk of occurrence of an adverse event, respectively. In some embodiments, low risk involves a lower risk compared to the detected level of proADM above the indicated value. In some embodiments, the high risk involves a higher risk compared to the detected level of proADM below the indicated value.

Bearing in mind the above, however, the determination of the high and/or low proADM severity levels is highly reliable in determining the presence or absence of subsequent adverse events when using the thresholds disclosed herein, such that the assessment of risk enables a medical professional to take appropriate action.

In some embodiments, a low or medium or high proADM severity level is indicative of the severity of the patient's physical condition relative to an adverse event.

The levels of pro-ADM in the sample may preferably be assigned to 3 different levels of proADM severity. High proADM levels indicate high severity levels, medium levels indicate medium severity levels and low levels indicate low severity levels. The respective concentrations of the threshold values determining the respective severity levels depend on a number of parameters, such as the sample isolation time point, e.g. after diagnosis and treatment of the patient has been initiated, and the method for determining the level of proADM or fragments thereof in said sample.

The threshold disclosed herein preferably refers to the measurement of the protein level of proADM or a fragment thereof in a plasma sample obtained from a patient by the sameifeier science and technology (Thermo Scientific) BRAHMS KRYPTOR assay. Accordingly, the values disclosed herein may vary to some extent depending on the detection/measurement method employed, and the specific values disclosed herein are also intended to read corresponding values determined by other methods.

In one embodiment of the invention, a low and/or medium severity level of proADM or one or more fragments thereof is indicative of an appropriate fluid treatment, wherein the low severity level is below a critical value in the range of 1.5nmol/l and 4 nmol/l. Any value within these ranges may be considered as a suitable threshold for a low severity level of proADM or a fragment thereof. For example, 1.5, 1.55, 1.6, 1.65, 1.7, 1.75, 1.8, 1.85, 1.9, 1.95, 2.0, 2.05, 2.1, 2.15, 2.2, 2.25, 2.3, 2.35, 2.4, 2.45, 2.5, 2.55, 2.6, 2.65, 2.7, 2.75, 2.8, 2.85, 2.9, 2.95, 3.0, 3.05, 3.1, 3.15, 3.2, 3.25, 3.3, 3.35, 3.4, 3.45, 3.5, 3.55, 3.6, 3.65, 3.7, 3.75, 3.8, 3.85, 3.9, 3.95, 4.0nmol/l.

In one embodiment of the invention, a high and/or medium severity level of proADM or one or more fragments thereof indicates a necessary decrease in volume of fluid administered, wherein the high severity level is above a critical value in the range of 6.5nmol/l to 12 nmol/l. Any value within these ranges may be considered as an appropriate threshold for a high severity level of proADM or a fragment thereof. For example, the number of the cells to be processed, 6.5, 6.55, 6.6, 6.65, 6.7, 6.75, 6.8, 6.85, 6.9, 6.95, 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, 7.4, 7.45, 7.5, 7.55, 7.6, 7.65, 7.7, 7.75, 7.8, 7.85, 7.9, 7.95, 8.0, 8.05, 8.1, 8.15, 8.2, 8.25, 8.3, 8.35, 8.4, 8.45, 8.5, 8.55, 8.6, 8.65, 8.7, 8.75, 8.8, 8.85, 8.9.9.95, 9.0, 9.05, 9.1, 9.15, 9.2, 9.25, 9.3, 9.35, 4.35, 8.3, 8.5, 8.55, 8.7, 8.8.7, 8.8.8.7, 8.75. 9.45, 9.5, 9.55, 9.6, 9.65, 9.7, 9.75, 9.8, 9.85, 9.9, 9.95, 10.0, 10.05, 10.1, 10.15, 10.2, 10.25, 10.3, 10.35, 10.4, 10.45, 10.5, 10.55, 10.6, 10.65, 10.7, 10.75, 10.8, 10.85, 10.9, 10.95, 11.0, 11.05, 11.1, 11.15, 11.2, 11.25, 11.3, 11.35, 11.4, 11.45, 11.5, 11.55, 11.6, 11.65, 11.7, 11.75, 11.8, 11.85, 11.9, 11.95, 12.0nmol/l.

All threshold values disclosed herein in relation to the level of a marker or biomarker (such as proADM or PCT) should be understood as "equal to or above" a certain threshold value or "equal to or below" a certain threshold value. For example, embodiments involving levels of proADM or one or more fragments thereof below 4nmol/l, preferably below 3nmol/l, more preferably below 2.7nmol/l should be understood to involve levels of proADM or one or more fragments thereof equal to or below 4nmol/l, preferably equal to or below 3nmol/l, more preferably equal to or below 2.7nmol/l. Conversely, an embodiment involving a level of proADM or one or more fragments thereof above 6.5nmol/l, preferably above 6.95nmol/l, more preferably above 10.9nmol/l should be understood to involve a level of proADM or one or more fragments thereof equal to or above 6.5nmol/l, preferably equal to or above 6.95nmol/l, more preferably equal to or above 10.9nmol/l.

According to another embodiment, the patient is an Intensive Care Unit (ICU) patient, wherein

-a level of proADM or one or more fragments thereof below a low severity level (threshold value) is indicative of a permission of the patient to leave the ICU, or

Levels of proADM or one or more fragments thereof at or above a high severity level (threshold) are indicative of modifying the treatment of patients in the ICU.

A particular advantage of the present invention is that the likelihood of a future adverse event occurring in the health of a patient can be assessed based on a classification of the determined level of proADM or one or more fragments thereof. Based on this evaluation, the following treatment options and decisions can be adjusted.

Treatment modification in the sense of the present invention will include, but is not limited to, adjusting the dosage or administration regimen of an ongoing drug, changing an ongoing treatment to a different treatment, adding additional treatment options to an ongoing treatment or stopping an ongoing treatment, or ascertaining and treating the cause of a dysfunction. Various treatments that may be applied to a patient in the context of the present invention have been disclosed in the detailed description of this patent application.

In a preferred embodiment of the invention, the method further comprises determining the level of one or more additional markers in the sample isolated from the patient.

The one or more additional markers may be determined in the same or different samples from the patient. In the case of different samples, the samples may be separated simultaneously with, before or after the separation of the samples to determine proADM or one or more fragments thereof. Whether the one or more additional markers are determined in the same sample or in different samples, the measurement may be performed in parallel with, simultaneously with, before and/or after the measurement of proADM.

In one embodiment, the one or more additional markers comprise PCT or one or more fragments thereof.

In one embodiment, at least one additional marker or clinical parameter (clinical score) is measured, preferably selected from the group comprising: procalcitonin, histone 3, histone 2A, histone 2B, histone 4 or one or more fragments thereof, C-reactive protein, (CRP), lactate, markers of coagulation system disorders, inflammatory cytokines (such as interleukins or chemokines), matrix metalloproteinases, qSOFA score, SOFA score, APACHE II score and SAPS II score.

The detection reagent for determining the level of proADM or one or more fragments thereof and optionally for determining the level of PCT or one or more fragments thereof and/or the additional markers of the invention is preferably selected from those necessary for performing the method, e.g. antibodies to proADM, suitable markers (such as fluorescent markers, preferably two separate fluorescent markers suitable for application in KRYPTOR assays), sample collection tubes.

In one embodiment of the methods described herein, the level of proADM or one or more fragments thereof and optionally further other biomarkers (such as, for example, PCT or one or more fragments thereof) is determined using a method selected from the group consisting of: mass Spectrometry (MS), luminescent Immunoassay (LIA), radioimmunoassay (RIA), chemiluminescent immunoassay and fluorescent immunoassay, enzyme Immunoassay (EIA), enzyme Linked Immunoassay (ELISA), luminescence-based bead array, magnetic bead-based array, protein microarray assay, rapid assay format (such as, for example, immunochromatographic strip test), rare hole complex assay and automated system/analyzer.

The method according to the invention may further be embodied as a homogeneous method, wherein the sandwich complex formed by the antibody/antibodies and the marker to be detected (e.g. proADM or a fragment thereof) is kept suspended in a liquid phase. In this case, it is preferred that when two antibodies are used, they are labeled with parts of the detection system, which results in the generation of a signal or triggering of a signal when the two antibodies are integrated into a single sandwich structure.

Such techniques should be embodied as fluorescence enhancement or fluorescence quenching detection methods. Particularly preferred aspects relate to the use of detection reagents which should be used in pairs, such as for example those described in US 4882733A, EP-B1 0180492 or EP-B1 0539477 and the prior art cited therein. In this way, it becomes possible to detect only the measurement of the reaction products directly comprising the two marker components in a single immunocomplex in the reaction mixture.

For example, such techniques are under the trade name(time-resolved amplification of the cave complex)Jet (Time Resolved Amplified Cryptate Emission)) or->Is provided to enable the teaching of the above application. Thus, in a particularly preferred aspect, the methods provided herein are performed using a diagnostic device. For example, the level of proADM protein or a fragment thereof and/or the level of any additional marker of the methods provided herein is determined. In a particularly preferred aspect, the diagnostic device is +. >

In one embodiment of the methods described herein, the method is an immunoassay, and wherein the assay is performed in homogeneous or heterogeneous phase.

In further embodiments of the methods described herein, the methods further comprise performing molecular analysis on a sample from the patient to detect the infection. The sample for molecular analysis to detect infection is preferably a blood sample. In a preferred embodiment, molecular analysis is a method aimed at detecting one or more biomolecules derived from a pathogen. The one or more biomolecules may be nucleic acids, proteins, sugars, carbohydrates, lipids and/or combinations thereof, such as glycosylated proteins, preferably nucleic acids. The biomolecules are preferably specific for one or more pathogens. According to a preferred embodiment, such biomolecules are detected by one or more methods for analyzing the biomolecules, selected from the group comprising nucleic acid amplification methods (such as PCR, qPCR, RT-PCR, qRT-PCR or isothermal amplification), mass spectrometry, enzymatic activity detection methods and immunoassay-based detection methods. Additional molecular analysis methods are known to those skilled in the art and are included in the methods of the invention.

In one embodiment of the method described herein, the first antibody and the second antibody are present in a form dispersed in a liquid reaction mixture, and wherein a first label component that is part of a fluorescence-or chemiluminescence-based extinction or amplification labeling system is bound to the first antibody and a second label component of the labeling system is bound to the second antibody, such that upon detection of binding of the two antibodies to the proADM or fragment thereof, a measurable signal is generated that allows detection of the resulting sandwich complex in a measurement solution.

In one embodiment of the methods described herein, the labeling system comprises a rare earth cryptate or chelate in combination with a fluorescent or chemiluminescent dye, particularly a cyanine-type dye.

In one embodiment of the methods described herein, the method further comprises comparing the determined level of proADM or one or more fragments thereof to a reference level, threshold value and/or population average value corresponding to proADM or fragments thereof in a control population (e.g., a healthy population), wherein the comparing is performed in a computer processor using computer executable code.

The method of the present invention may be partially computer-implemented. For example, the step of comparing the detected marker level (e.g., proADM or a fragment thereof) to a reference level may be performed in a computer system. In the computer system, the determined level of one or more markers may be combined with other marker levels and/or parameters of the subject to calculate a score indicative of diagnosis, prognosis, risk assessment, and/or risk stratification. For example, the determined values may be entered into the computer system (manually by a health professional or automatically from one or more devices for which one or more corresponding marker levels have been determined). Typically, the computer system stores values (e.g., marker levels or parameters (e.g., age, blood pressure, weight, gender, etc.) or clinical scoring systems (e.g., SOFA, qSOFA, BMI, PCT, platelet count, etc.) on computer readable media and calculates a score based on predefined and/or pre-stored reference levels or reference values.

In one embodiment of the invention, a software system may be employed in which machine learning algorithms are apparent, preferably to identify hospitalized patients at risk of sepsis, severe sepsis and septic shock using data from electronic medical records (EHRs). Machine learning methods can be trained on random forest classifiers using EHR data from patients (laboratory, biomarker expression, vital signs, and demographics). Machine learning is a type of artificial intelligence that enables computers to learn complex patterns in data without explicit programming, unlike simpler rule-based systems. Early studies used electronic medical record data to trigger alarms to detect clinical deterioration in general. In one embodiment of the invention, the processing of the proADM level may be incorporated into appropriate software for comparison with existing data sets, e.g. the proADM level may also be processed in machine learning software to assist in diagnosing or predicting the occurrence of adverse events.

The combined use of proADM or a fragment thereof with another biomarker, such as PCT or CRP, can be achieved in a single multiplex assay or in two separate assays performed on samples from patients. The samples may relate to the same sample or different samples. The assays used to detect and determine proADM and e.g. PCT may also be the same or different, e.g. immunoassays may be used to determine one of the above markers. A more detailed description of suitable assays is provided below.

The threshold and other reference levels of proADM or fragments thereof in a patient that has been diagnosed as critical and is being treated or is at risk of suffering from or suffering from a coagulation system disorder can be determined by the methods described previously. For example, methods for assessing variability of quantitative assays using coefficients of variation to establish reference and/or threshold values are known to the skilled artisan (George F. Reed et al, clinical and diagnostic laboratory immunology (Clin Diagn Lab Immunol), 2002;9 (6): 1235-1239).

In addition, functional assay sensitivity may be determined to indicate that a statistically significant value is used as a reference level or threshold according to established techniques. The laboratory is able to independently establish the functional sensitivity of the assay according to clinically relevant protocols. "functional sensitivity" may be considered as a concentration that produces a Coefficient of Variation (CV) (or some other predetermined CV%) of 20% and is therefore a measure of the precision of a measurement at low analyte levels. Thus, CV is a Standard Deviation (SD) normalization that allows for comparison of variability estimates over at least most of the operating range of the assay, regardless of the magnitude of the analyte concentration.

Furthermore, a method based on ROC analysis can be used to determine statistically significant differences between two clinical patient groups. The subject operating characteristic (ROC) curve measures the ranking efficiency of the fitting probabilities of the model to rank the response levels. The ROC curve may also help set standard points in diagnostic tests. The higher the diagonal curve, the better the fit. If the logical fit has more than two response levels, a generalized ROC curve will be generated. In this plot, there is a curve for each response level, which is the ROC curve for that level relative to all other levels. Software capable of performing such analysis to establish suitable reference levels and thresholds is available from, for example, JMP 12, JMP 13, statistical Discovery of the SAS.

The threshold value of PCT can be similarly determined. The literature is available to the skilled person for determining appropriate thresholds, e.g. Philipp Schuetz et al (BMC Medicine) 2011; 9:107) describe that PCT has a very high sensitivity to exclude infections at a threshold of 0.1 ng/mL. Terence Chan et al (molecular diagnostics Expert review (Expert Rev. Mol. Diagn); 2011;11 (5), 487.496) describe that indices calculated based on sensitivity and specificity, such as positive likelihood ratios and negative likelihood ratios, can also be used to evaluate the intensity of a diagnostic test. Values are typically plotted as a plurality of Critical Values (CVs) as a subject work characteristic. The area under the curve value is used to determine the optimal diagnosis-related CV. This document describes the variation of CV (critical value, which depends on the assay and study design) and a suitable method for determining the critical value.

population average levels of proADM or fragments thereof may also be used as reference values, e.g. average proADM population values, whereby patients diagnosed with a critical condition, such as patients with a coagulation system disorder, may be compared to a control population, wherein the control group preferably comprises more than 10, 20, 30, 40, 50 or more subjects.

In one embodiment of the invention, the threshold level of PCT in the serum sample may be a value in the range of 0.01ng/mL to 100.00ng/mL when measured using, for example, the Luminex MAC mepiquat electronic Bioscience Assay (Luminex MAC Pix E-Bioscience Assay) or the BRAHMS PCT-Kryptor.

In preferred embodiments, the critical level of PCT may be in the range of 0.01ng/mL to 100ng/mL, 0.05ng/mL to 50ng/mL, 0.1ng/mL to 20ng/mL, or 0.1ng/mL to 2ng/mL, and most preferably in the range of > 0.05ng/mL to 10 ng/mL. Any value within these ranges may be considered an appropriate threshold. For example, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100ng/mL may be employed. In some embodiments, the PCT level in healthy subjects is about 0.05ng/mL.

The advantages and embodiments of each of the various methods and kits of the invention disclosed herein are also applicable to and referred to in corresponding other methods and kits.

Embodiments of the invention relate to determining a method for therapy monitoring including health of a patient Prognosis, risk assessment and/or risk stratification of subsequent adverse events in a network

As described above, in some embodiments, the subsequent adverse event is one or more complications associated with fluid overload, sepsis, septic shock, organ failure, renal failure, organ dysfunction, and/or death.

The present invention relates to a method for therapy monitoring comprising prognosis, risk assessment and/or risk stratification of subsequent adverse events in the health of a patient, comprising

Providing a sample of the patient, wherein the patient has been diagnosed as critical and/or medical treatment has been started, wherein the sample is isolated from the patient after diagnosis and treatment has been started,

determining the level of proADM or one or more fragments thereof in said sample,

-wherein said level of proADM or one or more fragments thereof is related to the likelihood of a subsequent adverse event in the patient's health.

In one embodiment, the patient of the method of the invention has been diagnosed with a critical condition and has been undergoing treatment. Thus, the method of the present invention may be used to monitor the success of an already initiated treatment or therapy based on determining the likelihood of a subsequent adverse event. Therapy monitoring preferably involves prognosis of adverse events and/or risk stratification or risk assessment of patients with respect to future adverse events, wherein this risk assessment and determination of said risk should be considered as a means of monitoring the therapy initiated.

The physician or medical staff who is treating a patient who has been diagnosed as critical may employ the method of the invention in a different clinical setting, such as a primary care setting, or preferably in a hospital setting, such as in an emergency room or in an Intensive Care Unit (ICU). The method is very useful for monitoring the effect of an initiated therapy on critically ill patients and can be used to determine whether a patient under treatment is a high risk patient who should be under close medical observation and should likely receive additional therapeutic measures, or whether the patient is a low risk patient who may not need close observation and improved health of additional therapeutic measures, possibly because the initiated therapy successfully improves the patient's state. Initial treatment of critically ill patients can have a direct impact on the likelihood of adverse events in the patient's health. As such, assessment of risk/prognosis of future adverse events provides feedback or monitoring of initiated therapies.

The likelihood of a subsequent adverse event may be assessed by comparing the level of proADM or fragment thereof in the sample to a reference level (e.g., a threshold or critical value and/or population average value), wherein the reference level may correspond to proADM or fragment thereof in a healthy patient or a patient that has been diagnosed with a critical illness.

Thus, the method of the present invention may help predict the likelihood of a subsequent adverse event in the patient's health. This means that the method of the invention can distinguish between high risk patients who are more likely to suffer from complications or whose status will become more critical in the future, and low risk patients whose health condition is stable or even improved, so that the low risk patients are not expected to suffer from adverse events, such as patient death or worsening of clinical symptoms or signs of the patient, which may require certain therapeutic measures to be taken.

A particular advantage of the method of the invention is that patients identified as low risk patients by the method of the invention may be more quickly admitted to leave the ICU (typically a hospital) or may not need to be monitored as frequently. Also, for low risk patients, the intensity and/or frequency of observing the patient's health condition may be reduced. Thus, a hospital or other medical facility in charge of the patient can more efficiently decide which patients require rigorous medical care and observation. Thus, a corresponding hospital or institution may for example more efficiently hold high risk patients with ICU beds. This will lead to improved medical care for high risk patients, as medical staff may concentrate on such patients, while low risk patients may be admitted to leave the ICU. This would also be of significant benefit by avoiding the cost of unnecessary measures that would otherwise be applied to low risk patients.

The point in time at which the patient has been diagnosed as critical and the first therapeutic measure is initiated is defined as "point in time 0", which may be a reference for determining the sample isolation point in time of the proADM or a fragment thereof. If the patient diagnosis and the start of the treatment cannot be performed simultaneously, the time point 0 is the time point of the later event of the two events of the diagnosis and the start of the medical treatment. Typically, diagnosis of critically ill patients is immediately followed or accompanied by initiation of therapy.

It is entirely surprising that the level of proADM or a fragment thereof in a sample from a patient may provide critical information about the likelihood of a subsequent adverse event occurring in the health of said critically ill patient. There is no indication that a single measurement of proADM or a fragment thereof after diagnosis and initiation of treatment of critically ill patients can provide important information about the success of the ongoing treatment and prognosis of the health condition of the patient.

The use of proADM or a fragment thereof as a single parameter in embodiments of the invention is preferred over the use of other single parameters (such as biomarkers or clinical scores) because proADM is more accurate in predicting adverse events than: other markers, such as platelet count, PCT, CRP, lactate, or clinical scores (such as SOFA, SAPS II, or APACHE II); and markers of coagulation system disorders such as membrane microparticles, platelet count, average platelet volume (MPV), sCD14-ST, prothrombinase, antithrombin and/or antithrombin activity, cationic protein 18 (CAP 18), von willebrand factor (von Willebrand factor) (vWF) cleaving protease, lipoproteins in combination with CRP, fibrinogen, fibrin, B2GP1, GPIIb-IIIa, non-denatured fibrin D-dimer, platelet factor 4, histone and PT assays.

According to a preferred embodiment, the sample is separated from the patient during the medical examination.

According to a preferred embodiment, the sample is isolated from the patient within 30 minutes after the start of the diagnosis and treatment or at least 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 4 days, 7 days or 10 days after the start of the diagnosis and treatment. In other embodiments, samples are isolated from the patient 12-36 hours and/or 3-5 days after initiation of treatment.

The fact that the level of proADM or a fragment thereof at a certain point in time as short as about 30 minutes after the start of diagnosis and treatment can provide such information is completely unexpected.

In a preferred embodiment of the method of the invention, a sample is isolated from the patient about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 60 hours, 72 hours, 84 hours, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the diagnosis and treatment are initiated.

In other embodiments, samples are isolated at time points 30 minutes to 12 hours, 12-36 hours, 3-5 days, 7-14 days, 8-12 days, or 9-11 days after the diagnosis and beginning antibiotic treatment.

The time point at which the sample is obtained may be defined by a range between any of the above given values.

In another preferred embodiment of the invention, the patient has been diagnosed using at least one additional biomarker or clinical score. This is particularly advantageous in the context of the present invention if the initial diagnosis of the patient's significant disease at time point 0 is based at least in part on the level of at least one biomarker or a determined clinical score.

In certain embodiments, the invention includes determining additional parameters, such as markers, biomarkers, clinical scores, and the like.

In another preferred embodiment of the invention, the following has been used to diagnose a patient: biomarkers or clinical scores of at least one of Procalcitonin (PCT), lactate and C-reactive protein and/or clinical scores of at least one of qSOFA, SOFA, APACHE II, SAPS II, and markers of coagulation system disorders, such as membrane microparticles, platelet count, mean Platelet Volume (MPV), sCD14-ST, prothrombinase, antithrombin and/or antithrombin activity, cationic protein 18 (CAP 18), von willebrand factor (vWF) cleaving protease, lipoproteins in combination with CRP, fibrinogen, fibrin, B2GP1, GPIIb-IIIa, non-denatured fibrin D-dimer, platelet factor 4, histones and PT assays. In embodiments, the additional one or more markers comprise one or more histones. If the diagnosis of a patient is based on these markers, determining proADM or a fragment thereof in a sample of a patient that has been diagnosed as critically ill and is being treated proves to be particularly useful for therapy monitoring, since the prognosis of an adverse event in such a group of patients can be more accurate than in critically ill patients that have been diagnosed by other means.

In one embodiment of the invention, a critically ill patient is a patient diagnosed with, or at risk of developing, a coagulation system disorder, an infectious disease, a patient diagnosed with an infectious disease and one or more existing organ failure, a patient diagnosed with sepsis, severe sepsis or septic shock, and/or a post-traumatic or post-operative patient. In view of the data presented herein, the prognostic value of proADM in samples of these patient groups is particularly accurate in predicting the likelihood of adverse events for these patients.

In a preferred embodiment of the invention, the adverse event in the patient's health is death (preferably within 28-90 days after diagnosis and treatment initiation), new infection, organ failure and/or exacerbation of clinical symptoms requiring focal clearance, infusion of blood products, infusion of colloid, emergency surgery, invasive mechanical ventilation and/or renal or liver replacement.

In a preferred embodiment of the invention, said level of proADM or one or more fragments thereof is related to the likelihood of a subsequent adverse event in the health of said patient within 28 days after the start of diagnosis and treatment. In a further preferred embodiment of the invention said level of proADM or one or more fragments thereof is related to the likelihood of a subsequent adverse event in the health of said patient within 90 days after the start of diagnosis and treatment.

In certain embodiments of the invention, the treatment that the patient receives includes one or more of the following: antibiotic therapy, invasive mechanical ventilation, non-invasive mechanical ventilation, renal replacement therapy, use of vasopressors, fluid therapy, platelet infusion, blood transfusion, extracorporeal blood purification, and/or organ protection.

In a preferred embodiment of the invention, the sample is selected from the group consisting of a blood sample or a part thereof, a serum sample, a plasma sample and/or a urine sample.

In further embodiments of the invention, the level of proADM or one or more fragments thereof is related to the likelihood of a subsequent adverse event in the patient's health. In a preferred embodiment, the level of proADM or one or more fragments thereof is positively correlated with the likelihood of a subsequent adverse event in the health of the patient. In other words, the higher the determined proADM level, the greater the likelihood of a subsequent adverse event.

In accordance with a preferred embodiment of the present invention,

a low severity level of proADM or one or more fragments thereof, indicating the absence of or a low risk of a subsequent adverse event, wherein the low severity level is below 4nmol/l, preferably below 3nmol/l, more preferably below 2.7nmol/l, or

A high severity level of proADM or one or more fragments thereof, which is higher than 6.5nmol/l, preferably higher than 6.95nmol/l, more preferably higher than 10.9nmol/l, indicates a subsequent adverse event or indicates a higher risk of a subsequent adverse event.

In accordance with a preferred embodiment of the present invention,

levels of lower than 4nmol/l, preferably lower than 3nmol/l, more preferably lower than 2.7mol/l of proADM or one or more fragments thereof indicate no subsequent adverse events or a lower risk of indicating a subsequent adverse event, or

Levels of proADM or one or more fragments thereof above 6.5nmol/l, preferably above 6.95nmol/l, more preferably above 10.9mol/l are indicative of a subsequent adverse event or are at higher risk of indicating a subsequent adverse event.

In accordance with a preferred embodiment of the present invention,

-a low severity level of proADM or one or more fragments thereof indicates the absence of a subsequent adverse event, wherein the low severity level is below 2.7nmol/l, or

-a high severity level of proADM or one or more fragments thereof is indicative of a subsequent adverse event, wherein the high severity level is higher than 10.9nmol/l.

This embodiment of the invention is particularly advantageous when the level of proADM or a fragment thereof is determined on the day of diagnosis and treatment initiation of the patient, in particular in a sample isolated about 30 minutes after the diagnosis and treatment initiation.

In accordance with a preferred embodiment of the present invention,

A high severity level of proADM or one or more fragments thereof is indicative of a subsequent adverse event, wherein the high severity level is higher than 10.9nmol/l,

-wherein the level of proADM or one or more fragments thereof is preferably determined in a sample isolated on the day of diagnosis and treatment initiation.

In accordance with a preferred embodiment of the present invention,

-a low severity level of proADM or one or more fragments thereof indicates the absence of a subsequent adverse event, wherein the low severity level is below 2.8nmol/l, or

-a high severity level of proADM or one or more fragments thereof is indicative of a subsequent adverse event, wherein the high severity level is higher than 9.5nmol/l.

This embodiment of the invention is particularly advantageous when determining the level of proADM or a fragment thereof in a sample isolated 1 day after the start of said diagnosis and treatment.

In accordance with a preferred embodiment of the present invention,

A high severity level of proADM or one or more fragments thereof is indicative of a subsequent adverse event, wherein the high severity level is higher than 9.5nmol/l,

-wherein the level of proADM or a fragment thereof is preferably determined in a sample isolated 1 day after the start of diagnosis and treatment.

For example, if the level of proADM or a fragment thereof falls within the category of low severity level of proADM, the attending physician may more confidently decide to grant the patient to leave the ICU, as adverse events in the patient's health are preferably unlikely to occur within the next 28 days, more preferably within the next 90 days. Thus, it may not be necessary to leave this patient in the ICU. As assessed by a measurement of the risk of an adverse event, it may be concluded that the ongoing treatment is successfully improving the health of the patient.

Conversely, if a determination of the level of proADM or a fragment thereof in said ICU patient indicates a high severity level of proADM or a fragment thereof, the attending physician should leave the patient in the ICU. In addition, it should be considered to adjust the treatment of the patient, as the current treatment may not improve the health status of the patient, which is why the patient is more likely to suffer adverse events in the future.

According to a particularly preferred embodiment of the invention, the low severity level is below 2.75nmol/l, the sample is isolated from the ICU patient 1 day or more after the diagnosis and treatment is started and the low severity level of proADM or one or more fragments thereof indicates that the patient is admitted to leave the ICU.

The invention further relates to a method for therapy monitoring comprising prognosis, risk assessment and/or risk stratification of subsequent adverse events in the health of a patient, the method comprising

Providing a sample of the patient, wherein the patient is an Intensive Care Unit (ICU) patient and medical treatment has been started, wherein the sample is isolated from the patient after entering the ICU and treatment has been started,

In the context of the method of the invention involving ICU patients, the reference for determining the sample isolation time point of proADM or a fragment thereof is the time point at which the patient was admitted to the ICU and the first treatment measurement was started (time point 0). This time point corresponds to the diagnosis and treatment start time point in the method of the invention involving a patient who has been diagnosed with a critical disorder.

Thus, all embodiments of the method of the invention relating to a patient who has been diagnosed as critically ill are also considered to correspond to embodiments of the method of the invention relating to ICU patients.

Further relates to determining the presence of (or at the point in time of separation of) the first sample and the second sample Embodiments of the invention for the level or clinical scoring of PCT and/or other biomarkers of (c)

Preferred embodiments of the invention additionally comprise determining the level of PCT or one or more fragments thereof in a sample isolated from the patient. In a preferred embodiment, the sample used to determine the level of PCT or one or more fragments thereof is isolated before, at or after the point in time at which diagnosis and treatment are initiated.

It is particularly advantageous to combine the determination of proADM or a fragment thereof with the determination of PCT or a fragment thereof in a sample, wherein the sample used for determining proADM may be the same or a different sample used for detecting PCT.

The combination of the determination of proADM or a fragment thereof with the determination of PCT or a fragment thereof provides a synergistic effect in terms of accuracy and reliability in determining the risk of a subsequent adverse event, whether in the same sample or in samples obtained at different time points. These synergy also exist for the evaluation of proADM or fragments thereof in combination with other markers or clinical scores (e.g., platelet count, mean Platelet Volume (MPV), lactate, CRP, SOFA, SAPS II, APACHE II) or other clinical evaluations.

According to a further preferred embodiment of the invention, the method described herein additionally comprises

Determining the level of PCT or one or more fragments thereof in a first sample isolated from the patient, wherein the first sample is isolated before, at or after the point in time at which diagnosis and treatment begin,

-determining the level of PCT or one or more fragments thereof in a second sample isolated from the patient, wherein the second sample is isolated after the first sample, preferably within 30 minutes after isolating the first sample or 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 4 days, 7 days or 10 days after isolating the first sample, and

-determining a difference in the level of PCT or one or more fragments thereof in the second sample compared to the level of PCT or one or more fragments thereof in the first sample.

It is particularly advantageous to combine the determination of proADM or a fragment thereof in a sample isolated from a patient with the determination of PCT or a fragment thereof in a first sample and the determination of the level of PCT or a fragment thereof in a second sample isolated after the first sample, wherein the sample for determining proADM or a fragment thereof may be the same or different from the first sample or the second sample for determining PCT or a fragment thereof.

In a preferred embodiment, the methods described herein additionally comprise

Determining the level of PCT or one or more fragments thereof in a first sample isolated from the patient, wherein the first sample is isolated at or before the time point at which diagnosis and treatment is initiated (time point 0),

-determining the level of PCT or one or more fragments thereof in a second sample (sample according to claim 1) isolated from the patient after the start of diagnosis and treatment, preferably within 30 minutes after the start of diagnosis and treatment or 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 4 days, 7 days or 10 days after the start of diagnosis and treatment, and

It is particularly advantageous to combine the determination of proADM or a fragment thereof (in the second sample) with the determination of PCT or a fragment thereof in an earlier sample (the first sample) isolated from the patient and which can be used to diagnose the patient as critical at time point 0 and to determine the level of PCT or a fragment thereof in the sample isolated at a certain time point after the diagnosis and treatment have been started, which also preferably is the same time point at which proADM or a fragment thereof was determined. As indicated by the data below, determining the difference in the level of PCT or fragment thereof in the second sample compared to the first sample adds additional information to the information obtained from the level of proADM or fragment thereof in the second sample. Based on this combined information it is more likely that it is predicted whether an adverse event in the patient's health will occur, compared to the likelihood that an adverse event is predicted based on only information about the level of proADM or a fragment thereof in the second sample. This represents a surprising finding, as biomarkers of sepsis generally do not have synergy or complementarity, but only represent alternative diagnostic markers.

The preferred embodiment of the present invention additionally includes determining the SOFA. In a preferred embodiment, the SOFA is determined before, at, or after the point in time at which diagnosis and treatment begin.

It is particularly advantageous to combine the determination of proADM or a fragment thereof with the determination of SOFA, wherein the sample isolation time point for determining proADM may be the same as or different from the time point for determining SOFA.

Determining a first SOFA before, at or after the point in time when diagnosis and treatment begin,

-determining a second SOFA within 30 minutes after determining the first SOFA or within 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 4 days, 7 days or 10 days after determining the first SOFA, and

-determining a difference of the two determined SOFA's.

In a preferred embodiment, the methods described herein additionally comprise

Determining the SOFA at or before the point in time at which diagnosis and therapy begin (time 0),

-determining SOFA within 30 minutes after the start of the diagnosis and treatment or at least 30 minutes after the start of the diagnosis and treatment, preferably at least 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 4 days, 7 days or 10 days, and

-determining the difference of the SOFA determined after the diagnosis and treatment start from the SOFA determined at time point 0.

Preferred embodiments of the present invention additionally include determining SAPS II. In a preferred embodiment, SAPS II is determined before, at, or after the point in time at which diagnosis and treatment begin.

It is particularly advantageous to combine the determination of proADM or a fragment thereof with the determination of SAPS II, wherein the sample isolation time point for determining proADM may be the same or different from the time point for determining SAPS II.

Determining a first SAPS II before, at or after the point in time at which diagnosis and treatment begin,

-determining a second SAPS II within 30 minutes after determining the first SOFA or within 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 4 days, 7 days or 10 days after determining the first SAPS II, and

-determining a difference of the two determined SAPS II.

In a preferred embodiment, the methods described herein additionally comprise

Determining SAPS II at or before the time point of diagnosis and treatment initiation (time point 0),

-determining SAPS II within 30 minutes after the start of the diagnosis and treatment or at least 30 minutes after the start of the diagnosis and treatment, preferably at least 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 4 days, 7 days or 10 days, and

-determining the difference of SAPS II determined after the diagnosis and treatment start from SAPS II determined at time point 0.

Preferred embodiments of the present invention additionally include determining APACHE II. In a preferred embodiment, the APACHE II is determined before, at or after the point in time at which diagnosis and treatment begin.

It is particularly advantageous to combine the determination of proADM or a fragment thereof with the determination of APACHE II, wherein the sample isolation time point for determining proADM may be the same or different from the time point for determining APACHE II.

Determining a first APACHE II before, at or after the point in time at which diagnosis and therapy begin,

-determining a second APACHE II within 30 minutes after determining the first APACHE II or within 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 4 days, 7 days or 10 days after determining the first APACHE II, and

-determining a difference between the two determined APACHE II.

In a preferred embodiment, the methods described herein additionally comprise

Determining APACHE II at or before the point in time at which diagnosis and therapy begin (time 0),

-determining APACHE II within 30 minutes after the start of the diagnosis and treatment or at least 30 minutes after the start of the diagnosis and treatment, preferably at least 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 4 days, 7 days or 10 days, and

-determining the difference of APACHE II determined after the diagnosis and treatment start from APACHE II determined at time point 0.

In a preferred embodiment, high risk patients with increased PCT levels and higher severity levels of proADM or fragments thereof can be identified, which increased PCT levels and high severity levels represent a more accurate identification of such patients likely to suffer from adverse events in the future. Thus, treatment of such patients may be adjusted while minimizing the risk that such patients may be low risk patients.

The present invention relates to determining the level of proADM or one or more fragments thereof in a first sample and a second sample Examples

Preferred embodiments of the method of the invention additionally comprise

-determining the level of proADM or one or more fragments thereof in a first sample isolated from a patient, wherein the first sample is isolated before, at or after the point in time at which diagnosis and treatment are initiated, and

-determining the level of proADM or one or more fragments thereof in a second sample isolated from said patient, wherein said second sample is isolated after said first sample and after the point in time at which said diagnosis and treatment is started, preferably within 30 minutes after isolating said first sample or 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 4 days, 7 days or 10 days after isolating said first sample, and

-determining whether a difference in said level of proADM or one or more fragments thereof in said second sample compared to said level of proADM or one or more fragments thereof in said first sample is significant.

The first sample and the second sample for determining the level of proADM or one or more fragments thereof may be the same or different from the first sample and the second sample for determining the level of PCT or one or more fragments thereof.

Preferred embodiments of the method of the invention additionally comprise

Determining the level of proADM or one or more fragments thereof in a first sample isolated from the patient, wherein the first sample is isolated at or before the time point at which diagnosis and treatment is initiated (time point 0),

-determining the level of proADM or one or more fragments thereof in a second sample isolated after the start of the diagnosis and treatment, preferably within 30 minutes or 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 4 days, 7 days or 10 days after the start of the diagnosis and treatment, and

Preferred embodiments of the method of the invention additionally comprise

-determining the level of proADM or one or more fragments thereof in a first sample isolated from a patient, wherein the first sample is used to diagnose the patient as critical (time point 0), and

Further preferred embodiments of the method of the invention additionally comprise

-determining the level of proADM or one or more fragments thereof and optionally PCT or one or more fragments thereof in a first sample isolated from the patient, wherein the first sample is isolated at or before the time point of initiation of diagnosis and treatment (time point 0), and

-determining the level of proADM or one or more fragments thereof and optionally PCT or one or more fragments thereof in a second sample isolated from the patient after the start of the diagnosis and treatment, preferably within 30 minutes or at least 30 minutes after the start of the diagnosis and treatment, preferably 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 4 days, 7 days or 10 days after the start of the diagnosis and treatment, and

-determining a difference in said level of proADM or one or more fragments thereof and/or said level of PCT or a fragment thereof in said second sample compared to said level of proADM or one or more fragments thereof in said first sample.

-determining the level of proADM or one or more fragments thereof and optionally PCT or one or more fragments thereof in a first sample isolated from a patient, wherein the first sample is used to diagnose the patient as critical (time point 0), and

Surprisingly, determining a change in the level of proADM or a fragment thereof from the point in time at which diagnosis and treatment is started to a later point in time may provide additional information about the occurrence of future adverse events in the health of patients who have been diagnosed as critically ill, e.g. who have been diagnosed as suffering from thrombocytopenia. A great advantage of this embodiment of the invention is that the same sample used for determining the diagnostic marker at time point 0 can also be used for determining the baseline level of proADM or a fragment thereof, which can be compared with the level of proADM or a fragment thereof at a later time point after the diagnosis and treatment have been started. By determining changes in the level of proADM or a fragment thereof during patient treatment, the accuracy of predicting the occurrence of adverse events in the health of a patient can be further improved.

In one embodiment of the methods described herein, an elevated level of proADM or one or more fragments thereof in the second sample as compared to the first sample is indicative of a subsequent adverse event.

Surprisingly, based on a change in the level of proADM or a fragment thereof, the likelihood of occurrence of an adverse event in the health of a patient can be reliably predicted without the need to determine additional markers. An increase in the level or severity of proADM or a fragment thereof from the point in time at which diagnosis and treatment is initiated indicates that adverse events may occur. Thus, based on the change in proADM or fragments thereof during the course of treatment, the physician can decide whether to change or modify the treatment of the patient or to adhere to the initial treatment.

In a preferred embodiment of the method of the invention,

-an elevated level of proADM or one or more fragments thereof and an elevated level of PCT or one or more fragments thereof in the second sample compared to the first sample is indicative of a subsequent adverse event, and/or

In some embodiments of the invention, the elevated level of proADM or a fragment thereof in the second sample as compared to the first sample relates to an elevated severity level of proADM or a fragment thereof. Conversely, in some embodiments of the invention, a lower level of proADM or a fragment thereof in the second sample as compared to the first sample refers to a lower severity level of proADM or a fragment thereof in the second sample as compared to the first sample.

A great advantage is that in connection with a determined change in PCT or a fragment thereof based on a change in the level of proADM or a fragment thereof during the treatment of critically ill patients, the likelihood of an adverse event in the health of the patient can be assessed. Thus, high risk patients and low risk patients can be reliably identified based on the changes in the two markers.

Advantageously, by performing a combined analysis of the level of PCT or fragment thereof and the change in severity level of proADM or fragment thereof at the time point (later time point) when the second sample was isolated in the ICU patient, it can be decided whether the patient is a low risk patient who can be admitted to leave the ICU while maintaining ongoing treatment, or whether the patient is a high risk patient who needs to modify or adjust the current treatment at the ICU to prevent the occurrence of adverse events indicated by the corresponding combination of PCT level change and current severity level of proADM.

In a further embodiment, the invention relates to a kit for performing the method of the invention, wherein the kit comprises

A detection reagent for determining the level of proADM or one or more fragments thereof and optionally additionally for determining PCT levels, and

-reference data, such as a reference level, corresponding to a high and/or low severity level of proADM, wherein the low severity level is below 4nmol/l, preferably below 3nmol/l, more preferably below 2.7nmol/l, and the high severity level is above 6.5nmol/l, preferably above 6.95nmol/l, more preferably above 10.9nmol/l, and optionally PCT, wherein the reference data is preferably stored on a computer readable medium and/or used in the form of computer executable code configured to compare the determined level of proADM or one or more fragments thereof and optionally the determined level of further PCT, lactate and/or C-reactive protein or one or more fragments thereof with the reference data.

-a detection reagent for determining the level of proADM or one or more fragments thereof and optionally the level of one or more markers of PCT and/or coagulation system disorders, such as membrane particles, platelet count, mean Platelet Volume (MPV), sCD14-ST, prothrombinase, antithrombin and/or antithrombin activity, cationic protein 18 (CAP 18), von willebrand factor (vWF) cleaving protease, lipoproteins in combination with CRP, fibrinogen, fibrin, B2GP1, GPIIb-IIIa, non-denatured fibrin D-dimer, platelet factor 4, histones and PT assays, in a sample from a subject, and

reference data, such as reference levels, corresponding to a high and/or low severity level of proADM, wherein the low severity level is below 4nmol/l, preferably below 3nmol/l, more preferably below 2.7nmol/l, and the high severity level is above 6.5nmol/l, preferably above 6.95nmol/l, more preferably above 10.9nmol/l, and optionally one or more markers of PCT and/or coagulation system disorders, such as membrane particles, platelet count, average platelet volume, sCD14-ST, prothrombinase, antithrombin and/or antithrombin activity, cationic protein 18 (CAP 18), von willebrand factor (vWF) cleaving protease, lipoproteins in combination with CRP, fibrinogen, fibrin, B2GP1, ib-IIIa, non-denatured fibrin D-dimer, platelet factor 4, histones and-assays, wherein the reference data is preferably stored on a computer readable medium and/or used in the form of computer executable code configured to compare the determined level of proADM or one or more fragments thereof and optionally the determined level of PCT and/or the determined level of one or more markers of coagulation system disorders, such as membrane particles, platelet count, average platelet volume (MPV), sCD14-ST, prothrombinase, antithrombin and/or antithrombin activity, cationic protein 18 (CAP 18), von willebrand factor (vWF) cleaving protease, lipoproteins in combination with CRP, with the reference data, fibrinogen, fibrin, B2GP1, GPIIb-IIIa, non-denatured fibrin D-dimer, platelet factor 4, histone and PT assays.

-a detection reagent for determining the level of proADM or one or more fragments thereof and optionally additionally for determining the level of PCT or one or more fragments thereof in a sample from a subject, and

-reference data, such as a reference level, corresponding to a high and/or low severity level of proADM, wherein the low severity level is below 4nmol/l, preferably below 3nmol/l, more preferably below 2.7nmol/l, and the high severity level is above 6.5nmol/l, preferably above 6.95nmol/l, more preferably above 10.9nmol/l, and optionally PCT level, wherein the reference data is preferably stored on a computer readable medium and/or used in the form of computer executable code configured to compare the determined level of proADM or one or more fragments thereof, and optionally the determined level of PCT or one or more fragments thereof, with the reference data.

In one embodiment, the invention relates to a kit for performing the method described herein, comprising:

-reference data, such as a reference level, corresponding to a proADM severity level according to claim 6 and/or 9, and optionally a PCT level, wherein the reference data is preferably stored on a computer readable medium and/or used in the form of computer executable code configured to compare a determined level of proADM or one or more fragments thereof and optionally additionally PCT or one or more fragments thereof with the reference data.

Detailed Description

The present invention is based on methods of therapy guidance, stratification and/or monitoring of fluid therapy based on adrenomedullin precursor (proADM) levels.

As used herein, the term "fluid therapy" refers to the administration of a liquid to a subject in need thereof. The term "fluid replenishment" or "fluid resuscitation" may also be used. Fluid therapy includes, but is not limited to, supplementing fluid with oral rehydration therapy (hydration), intravenous therapy, rectally, or injecting fluid into subcutaneous tissue. Intravenous methods are preferred.

The term "prescription of fluid therapy" includes any indication, information, and/or description regarding the fluid therapy to be administered, including but not limited to the volume, dosage, rate, mode and/or type of fluid to be administered. The decision as to which fluids are suitable for each patient depends on the disease, the type of fluid lost and the body compartment or compartments that require additional capacity. The skilled person is able to make these evaluations based on the indications provided herein. Where applicable, the renal function, cardiac function, blood gas and electrolyte levels of the patient may be considered.

In one embodiment, the present invention relates to prescribing for fluid resuscitation, fluid maintenance, fluid replenishment, and/or fluid redistribution.

A "colloid" is a dispersion of large organic molecules (e.g., gelofusin, voluven). Colloids are generally suspensions of molecules in a carrier solution that, due to their molecular weight, are relatively unable to cross healthy semipermeable capillary membranes. In one embodiment, the method indicates that the fluid therapy comprises a colloid or blood selected from gelatin, albumin and/or starch solutions or a blood-derived fluid.

The nature of colloidal infusion is largely dependent on its molecular size. Many modern colloidal solutions are based on hydroxyethyl starch (HES) that has a high molecular weight (70,000-450,000 daltons) and can provide a volume expansion of 6-24 hours. The duration of action of the solution depends on the molecular size of its starch (macromolecules tend to have a longer duration), the rate of degradation and the permeability of the vascular endothelium. Tetra starch (40% substituted HES) with an average molecular weight of 130,000 daltons was active for 4-6 hours. The molecular weight of the modified fluid gelatin derived from animal collagen was 30,000 daltons. The effective half-life of the fluid gelatin is about 4 hours, but its capacity recovery in patients with capillary leakage can be shorter.

Without being limited by theory, colloids generally allow for rapid recovery of intravascular volume. The use of albumin is performed with reduced synthesis of coagulation factors (e.g., severe liver failure). The lower overall capacity load necessary to apply the colloid is often seen as an advantage. Regarding the capacity of colloids to replenish the intravascular volume, it is generally assumed that 3L of the crystal solution corresponds to 1L of the colloid solution. However, other studies reported a ratio of saline to albumin of 1.4 L:1L.

Gelatin infusion is similar in molecular size to albumin and thus may not allow for a significant reduction in volume administered. It may be possible to use smaller volumes of large starch molecule solutions (e.g., voluven) to supplement intravascular volume. In particular, where epithelial wall permeability is increased (e.g., sepsis, other inflammatory conditions, anesthesia), starch may be more effective in reducing leakage into the interstitial space by increasing the oncotic pressure.

"crystals" are aqueous solutions of small molecules (e.g., sodium chloride, glucose, hartmann's solution, or Ringer's solution). The crystals are typically solutions that are freely permeable but contain ions of sodium and chloride at concentrations that determine the tonicity of the fluid.

Guidance regarding fluid therapy is available to the skilled artisan, as in "intravenous fluid therapy: intravenous fluid therapy (Intravenous Fluid Therapy: intravenous Fluid Therapy in Adults in Hospital) for hospitalized adults; NICE clinical guidelines (NICE Clinical Guidelines), no. 174.

"fluid resuscitation" preferably refers to the emergency administration of IV fluid to restore circulation to vital organs after loss of vascular content, plasma loss, excessive external fluid and electrolyte loss (typically from the Gastrointestinal (GI) tract) or severe internal loss (e.g., from fluid redistribution in sepsis) due to bleeding. Fluid resuscitation is required in the event of acute circulatory shock or insufficient intravascular volume. The aim is to restore circulatory capacity and to increase cardiac output, thereby restoring tissue perfusion and oxygen delivery. In the case of resuscitation, restoration of intravascular volume is initially important, and any sodium or colloid based fluid may be used to do so.

"routine fluid maintenance" preferably refers to the administration of IV fluid to a patient who is not able to meet their normal fluid or electrolyte requirements at all by oral or enteral routes, but is otherwise good in terms of fluid and electrolyte balance and handling, i.e., the patient is substantially normoglycemic, without significant depletion, sustained abnormal loss or redistribution problems. However, even when prescribing IV fluids for more complex cases, there is still a need to meet the patient's regular maintenance needs, adjusting the maintenance prescription to account for more complex fluid or electrolyte issues.

"fluid replenishment" preferably refers to administration of IV fluid to treat losses from intravascular and/or other fluid compartments, but is not urgently needed for resuscitation, but is still needed to correct existing moisture and/or electrolyte depletion or sustained external losses. These losses usually come from the GI or urinary tract, although high, non-sensible losses occur when febrile, and burn patients may lose high volumes of substances, effectively plasma.

"fluid redistribution" preferably refers to fluid handling that accounts for significant internal fluid distribution changes or anomalies in addition to external fluid and electrolyte losses. This type of problem is particularly seen in patients with sepsis, other severe conditions, major surgery or with major heart, liver or kidney co-diseases. Many of these patients develop oedema caused by excess sodium and water and some barrier fluid in the GI tract or the chest/peritoneal cavity.

The most suitable fluid and electrolyte application method is the simplest, safest and most effective. Where possible, the oral route should be used, whereas IV fluids are generally avoided in patients who are eating and drinking water. If safe oral intake is impaired, but there is GI function available in the enteral tube, the possibility of enteral tube administration should also be considered.

Many different crystal solutions, artificial colloid solutions, and albumin solutions are available for IV fluid therapy. The objective is to meet an estimate of the total fluid and electrolyte requirements. It is a theoretical advantage to administer colloid rather than crystals when resuscitating a patient with low blood volume, as colloid-based fluids typically remain in circulation for a longer period of time. Crystals are distributed throughout the ECF and the traditional teaching is that the effect of crystal infusion on plasma volume is relatively limited and transient.

Without limitation, therapeutic fluids include, but are not limited to

Isotonic saline: sodium chloride 0.9% with or without additional potassium is one of the most commonly used IV fluids. As with all crystals, 0.9% sodium chloride was distributed throughout the ECF, and the effect of infusion on plasma volume was generally more transient than that of the colloid. Traditionally, 0.9% sodium chloride infusion is considered to be one quarter to one third of the volume of an infusion to expand blood volume only, the remainder being sequestered in the interstitial space.

Equilibrium crystal solution: the equilibrium crystals are also distributed throughout the ECF and thus have a similar efficacy in terms of plasma volume expansion as 0.9% sodium chloride. However, the equilibrium crystal does have theoretical advantages because it contains slightly less sodium and significantly less chlorine, and it may already have some potassium, calcium and magnesium content. In cases where significant acidosis is required, which is common in resuscitation, an equilibration solution containing lactate or other buffers may also provide advantages.

Glucose and dextrose-saline: solutions such as 5% glucose and glucose/saline with or without potassium are not used to revive or replace the loss of electrolyte-rich. However, it is a useful means of providing free water once glucose is metabolized, primarily through systemic water distribution, with very limited and transient effects on blood volume.

And (3) synthesizing a colloid: synthetic colloids such as HES contain fluid dispersed amorphous molecules or super microscopic particles that are typically crystalline. The colloidal particles are large enough that they should remain within the cycle and thus apply an expansion pressure across the capillary membrane. Some formulations of hydroxyethyl starch are suspended in 0.9% sodium chloride, while others are suspended in an equilibrium solution that should make them more 'physiological'. Most of the semisynthetic colloids currently available, such as HES, contain 140-154mmol of sodium that can promote positive sodium balance in heavier patients in the same way as 0.9% sodium chloride, although colloids do generally contain less chlorine.

Albumin solution: as with synthetic colloids, infusion of albumin solutions may theoretically be potentially beneficial from a better intravascular volume expansion, albeit at a high cost. Concentrated (20-25%) low sodium albumin can also be valuable in terms of fluid redistribution problems, especially when edema caused by total sodium and water overload occurs in patients with low plasma volumes following critical illness or injury.

Gelatin solution: gelatin is often administered as a plasma volume replacement. This means that it supplements the fluid lost from the circulation. A common and commercially available liquid gelatin is jiale, which is a 4% w/v succinylated gelatin (also known as modified fluid gelatin) solution used as an intravenous colloid that behaves much like albumin-filled blood.

Compared with the traditional method, the invention has the following advantages: the method and the kit are rapid, objective, convenient to use and accurate for therapy monitoring. The methods and kits of the invention relate to markers and clinical scores that can be readily measured in hospital routine methods, as the levels of proADM, PCT, lactate, c-reactive protein, SOFA, APACHE II, SAPS II, etc. can be determined in a conventionally obtained blood sample, additional biological fluid, or a sample obtained from a subject.

As used herein, a "patient" or "subject" may be a vertebrate. In the context of the present invention, the term "subject" encompasses both humans and animals, in particular mammals, as well as other organisms.

In the context of the present invention, an "adverse event in the health of a patient" relates to an event that indicates a complication of the patient or a worsening of the health condition of the patient. Such adverse events include, but are not limited to, patient death within 28-90 days after diagnosis and treatment initiation, occurrence of infection or new infection, organ failure (e.g., renal failure), and general clinical signs or symptoms of the patient worsening, such as hypotension or hypertension, tachycardia or bradycardia, coagulation system disorders, disseminated intravascular coagulation, abnormal platelet levels, thrombocytopenia and organ dysfunction or organ failure associated with thrombocytopenia.

Furthermore, examples of adverse events include cases where exacerbation of a clinical symptom indicates a need to take therapeutic measures such as focal clearance, infusion of blood products, infusion of colloid, invasive mechanical ventilation, platelet infusion, non-invasive mechanical ventilation, emergency surgery, organ replacement therapies (such as kidney or liver replacement), and vasopressors. In addition, adverse events may include providing corticosteroids, blood transfusion or platelet infusion, infusion of blood components (such as serum, plasma or specific cells or combinations thereof), drugs that promote platelet formation, etiologic treatment, or performing splenectomy.

Patients who have been diagnosed as "critical" as described herein may be diagnosed as Intensive Care Unit (ICU) patients, patients who need to have their health status continuously and/or closely observed, patients diagnosed as having sepsis, severe sepsis or septic shock, patients diagnosed as having infectious disease and one or more existing organ failure, pre-or post-operative patients, post-traumatic patients, trauma patients (e.g., accident patients, burn patients), patients with one or more open lesions. The subject described herein may be in an emergency room or intensive care unit or in other point-of-care settings (e.g., in an emergency transport facility such as an ambulance) or at a general practitioner who is faced with a patient with the symptoms. Furthermore, in the context of the present invention, critical conditions may refer to a patient being at risk of suffering from or suffering from a coagulation system disorder. Thus, in the context of the present invention, critical conditions preferably refer to a patient at risk of suffering from or suffering from a low number of platelets (thrombocytopenia).

The term "ICU patient" relates to, but is not limited to, a patient who has received treatment in an intensive care unit. An intensive care unit may also be referred to as an intensive care unit or intensive care unit (ITU) or Critical Care Unit (CCU), a special department of a hospital or healthcare facility that provides intensive care medicine. ICU patients often suffer from serious and life threatening diseases and injuries that require continuous close monitoring and support from professional equipment and medications in order to ensure proper physical functioning. Common conditions treated within the ICU include, but are not limited to, acute or Adult Respiratory Distress Syndrome (ARDS), trauma, organ failure and sepsis.

As used herein, "diagnosis" in the context of the present invention relates to the identification and (early) detection of clinical conditions in a subject associated with an infectious disease. The term "diagnosis" may also encompass the assessment of the severity of an infectious disease.

"prognosis" refers to the prediction of the efficacy or specific risk of an infectious disease-based subject. This may also include an estimate of the likelihood of rehabilitation or likelihood of poor efficacy of the subject.

The method of the invention can also be used for monitoring. "monitoring" involves tracking established infectious diseases, disorders, complications or risks, for example, to analyze the progression of a disease or the effect of a particular treatment or therapy on the disease progression of critically ill patients or the infectious disease of patients.

In the context of the present invention, the term "therapy monitoring" or "therapy control" refers to monitoring and/or adjusting the therapeutic treatment of the subject, e.g. by obtaining feedback on the efficacy of the therapy.

In the present invention, the terms "risk assessment" and "risk stratification" relate to the classification of a subject into different risk groups according to the further prognosis of the subject. Risk assessment also involves layering for application of preventive and/or therapeutic measures. Examples of risk stratification are low risk levels, medium risk levels, and high risk levels as disclosed herein.

As used herein, the term "therapy guidance" refers to the application of certain therapies or medical interventions based on one or more biomarkers and/or values of clinical parameters and/or clinical scores.

It is to be understood that in the context of the present invention, "determining the level of proADM or one or more fragments thereof" etc. refers to any means of determining proADM or fragments thereof. Fragments may be of any length, for example at least about 5, 10, 20, 30, 40, 50 or 100 amino acids, as long as the fragments allow for a clear determination of the level of proADM or fragments thereof. In a particularly preferred aspect of the invention, "determining the level of proADM" refers to determining the level of the middle adrenomedullin precursor (MR-proADM). MR-proADM is a fragment and/or region of proADM.

The peptide Adrenomedullin (ADM) was found to be a antihypertensive peptide comprising 52 amino acids isolated from human pheochromocytoma (Kitamura et al, 1993). Adrenomedullin (ADM) is encoded as a precursor peptide comprising 185 amino acids ("adrenomedullin prepro" or "pre proADM"). SEQ ID NO: an exemplary amino acid sequence of proADM is given in 1.

SEQ ID NO:1: amino acid sequence of pre-pro-ADM:

1 MKLVSVALMY LGSLAFLGAD TARLDVASEF RKKWNKWALS RGKRELRMSS

51 SYPTGLADVK AGPAQTLIRP QDMKGASRSP EDSSPDAARI RVKRYRQSMN

101 NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSK ISPQGYGRRR

151 RRSLPEAGPG RTLVSSKPQA HGAPAPPSGS APHFL

ADM comprises positions 95-146 of the pre-proADM amino acid sequence and is a splice product thereof. "adrenomedullin precursor" ("proADM") refers to pre-proADM without a signal sequence (amino acids 1 to 21), i.e. to amino acid residues 22 to 185 of pre-proADM. "middle adrenomedullin precursor" ("MR-proADM") refers to amino acids 42 to 95 of pre-proADM. SEQ ID NO:2, an exemplary amino acid sequence of MR-proADM is given.

SEQ ID NO:2: amino acid sequence of MR-pro-ADM (AS 45-92 of pre-pro-ADM):

ELRMSSSYPT GLADVKAGPA QTLIRPQDMK GASRSPEDSS PDAARIRV

it is also contemplated herein that peptides of pre-proADM or MR-proADM and fragments thereof may be used in the methods described herein. For example, the peptide or fragment thereof may comprise amino acids 22-41 of pre-proADM (PAMP peptide) or amino acids 95-146 of pre-proADM (mature adrenomedullin, including biologically active forms, also known as bio-ADM). The C-terminal fragment of proADM (amino acids 153 to 185 of proADM) is called epinephrine. Fragments of proADM peptides or MR-proADM may comprise, for example, at least about 5, 10, 20, 30 or more amino acids. Thus, a fragment of proADM may for example be selected from the group consisting of MR-proADM, PAMP, epinephrine and mature adrenomedullin, preferably in this context the fragment is MR-proADM.

Determination of these different forms of ADM or proADM and fragments thereof also encompasses measuring and/or detecting specific sub-regions of these molecules, for example by employing antibodies or other affinity reagents directed against specific parts of the molecules, or by determining the presence and/or amount of the molecules by measuring a part of the protein by means of mass spectrometry.

Any one or more of the "ADM peptides or fragments" described herein may be employed in the present invention.

The methods and kits of the invention may further comprise determining at least one additional biomarker, marker, clinical score and/or parameter other than proADM.

As used herein, a parameter is a factor that may be helpful in defining a characteristic, feature, or measurable of a particular system. Parameters are important factors for health and physiological related assessment, such as disease/disorder/risk of clinical pathology, preferably one or more organ dysfunction. Furthermore, a parameter is defined as a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to therapeutic interventions. Exemplary parameters may be selected from the group consisting of: acute physiology and chronic health assessment II (APACHE II), simplified acute physiology score (sapsi II score), sequential organ failure assessment score (SOFA score), rapid sequential organ failure assessment score (qSOFA score), body mass index, body weight, age, sex, IGS II, fluid intake, white blood cell count, sodium, platelet count, average platelet volume (MPV), potassium, temperature, blood pressure, dopamine, bilirubin, respiration rate, oxygen partial pressure, worldwide congress of neurosurgery (WFNS) classification, and glasgow coma index (GCS).

As used herein, terms such as "marker," "surrogate," "prognostic marker," "factor" or "biomarker" or "biological marker" are used interchangeably and relate to a measurable and quantifiable biological marker (e.g., the presence of a particular protein or enzyme concentration or fragment thereof, a particular hormone concentration or fragment thereof, or a biological substance or fragment thereof) that serves as an index for health and physiological related assessment, such as disease/disorder/risk of clinical condition, preferably an adverse event. A marker or biomarker is defined as a characteristic that can be objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to therapeutic interventions. Biomarkers can be measured in a sample (e.g., blood, plasma, urine, or tissue test).

The at least one additional marker and/or parameter of the subject may be selected from the group consisting of: lactate levels in the sample, procalcitonin (PCT) levels in the sample, sequential organ failure assessment scores (SOFA scores) (optionally, rapid SOFA score), simplified Acute Physiology Score (SAPSII) of the subject, acute physiology and chronic health assessment II (APACHE II) score of the subject, and soluble fms-like tyrosine kinase-1 (sFlt-1) levels, histone H2A, histone H2B, histone H3, histone H4, calcitonin, endothelin-1 (ET-1), arginine Vasopressin (AVP), atrial Natriuretic Peptide (ANP), neutrophil gelatinase-associated lipocalin (NGAL), troponin, brain Natriuretic Peptide (BNP), C-reactive protein (CRP), pancreatic Dan Danbai (PSP), myeloid cell expressed trigger receptor-1 (TREM 1), interleukin 6 (IL-6), interleukin-1, interleukin 24 (IL-24), interleukin 22 (IL-22), interleukin (IL-20), other IL, presen (sCD 14-), lipopolysaccharide Binding Protein (LBP), alpha-1-antitrypsin, matrix metalloproteinase 2 (MMP 2), MMP2 (MMP 8), MMP matrix metalloproteinase (MMP 9), MMP9 (MMP 9, and factor (MMP-37 a) growth factor (MMP 9), S100B protein and tumor necrosis factor alpha (TNF alpha), neopterin, alpha-1-antitrypsin, arginine vasopressin (AVP, proAVP or pepsin), procalcitonin, atrial natriuretic peptide (ANP, pro-ANP), endothelin-1, CCL1/TCA3, CCL11, CCL12/MCP-5, CCL13/MCP-4, CCL14, CCL15, CCL16, CCL17/TARC, CCL18, CCL19 CCL2/MCP-1, CCL20, CCL21, CCL22/MDC, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3L3, CCL4L1/LAG-1, CCL5, CCL6, CCL7, CCL8, CCL9, CX3CL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CXCL2/MIP-2 CXCL3, CXCL4, CXCL5, CXCL6, CXCL7/Ppbp, CXCL9, IL8/CXCL8, XCL1, XCL2, FAM19A1, FAM19A2, FAM19A3, FAM19A4, FAM19A5, CLCF1, CNTF, IL11, IL31, IL6, leptin, LIF, OSM, IFNA1, IFNA10, IFNA13, IFNA14, IFNA2, IFNA4, IFNA7, IFNB1, IFNE, IFNG, IFNZ, IFNA8, IFNA5/IFNaG, IFomega/IFNW 1 BAFF, 4-1BBL, TNFSF8, CD40LG, CD70, CD95L/CD178, EDa-A1, TNFSF14, LTA/TNFB, LTB, TNFa, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF15, TNFSF4, IL18BP, IL1A, IL1B, IL F10, IL1F3/IL1RA, IL1F5, IL1F6, IL1F7, IL1F8, IL1RL2, IL1F9, IL33 or fragments thereof. Additional markers include membrane microparticles, platelet count, average platelet volume (MPV), sCD14-ST, prothrombinase, antithrombin and/or antithrombin activity, cationic protein 18 (CAP 18), von Willebrand factor (vWF) cleaving protease, lipoproteins in combination with CRP, fibrinogen, fibrin, B2GP1, GPIIb-IIIa, non-denatured fibrin D-dimer, platelet factor 4, histone and PT assays.

As used herein, "procalcitonin" or "PCT" refers to a peptide spanning amino acid residues 1-116, 2-116 or 3-116 of a procalcitonin peptide or a fragment thereof. PCT is a peptide precursor of the hormone calcitonin. Thus, the procalcitonin fragment is at least 12 amino acids in length, preferably more than 50 amino acids, more preferably more than 110 amino acids. PCT may include post-translational modifications such as glycosylation, lipidation or derivatization. Procalcitonin is a precursor of calcitonin and procalcitonin. Thus, under normal conditions, PCT levels in the circulation are very low (< about 0.05 ng/ml).

PCT levels in a sample of a subject can be determined by an immunoassay as described herein. As used herein, the level of ribonucleic acid or deoxyribonucleic acid encoding a "procalcitonin" or "PCT" can also be determined. Methods for determining PCT are known to the skilled person, for example by using the product obtained from the company sammer feier technology (Thermo Fisher Scientific)/bolames limited (b·r·a·h·m·s GmbH).

It is understood that "determining the level of at least one histone" and the like refers to determining the level of at least one histone or a fragment of said at least one histone in a sample. Specifically, the level of histone H2B, H4, H2A and/or H4 in the sample is determined. Thus, the at least one histone determined in the sample may be a free histone, or the at least one histone determined in the sample may be present and may be assembled in a macromolecular complex, such as an octamer, a nucleosome and/or NET.

In particular aspects of the invention, the level of histone proteins or fragments thereof that are not assembled in macromolecular complexes such as nucleosomes, octamers, or Neutrophil Extracellular Traps (NET) can be determined in a sample. One or more such histones are referred to herein as "free histone(s)". Thus, the level of the at least one histone protein may in particular be the level of at least one free histone protein.

The level of such free histones can be determined by detecting amino acid sequences or structural epitopes of histones that are not accessible in the assembled stoichiometric macromolecular complexes such as the mononucleosomes or octamers. In such structures, specific regions of histone are covered and thus are not spatially accessible as shown for neutrophil extracellular traps ("NET"). In addition, in octamers or nucleosomes, regions of histones are also involved in intramolecular interactions, such as interactions between histones alone. Thus, the region/peptide/epitope of a histone protein determined in the context of the present invention can determine whether the histone protein is free histone or assembled in a macromolecular complex. For example, in an immunoassay-based method, when a histone (e.g., H4) is part of the octamer core of a nucleosome, the antibodies utilized may not detect the histone because the epitope is structurally unreachable. The regions/peptides/epitopes of histones that can be used to determine free histones are exemplified herein below. For example, the region/peptide/epitope of the N-terminal or C-terminal tail of a histone can be used to determine the histone irrespective of whether the histone is assembled in a macromolecular complex or the free histone according to the invention.

"stoichiometric" in this context refers to an intact complex, such as a mononuclear cell or octamer. "free histone" may also include non-chromatin binding histones. For example, "free histone" may also include histone or non-octameric histone complexes alone. The free histones may be bound (e.g., transiently) to individual histones, e.g., the histones may form homodimers or heterodimers. Free histones may also form homotetramers or heterotetramers. Homotetramers or heterotetramers may consist of four molecules of histone proteins, e.g., H2A, H2B, H3 and/or H4. Typical heterotetramers are formed by two heterodimers, each of which consists of H3 and H4. It is also understood herein that hetero-tetramers may be formed by H2A and H2B. It is also contemplated herein that the heterotetramers may be formed by one heterodimer consisting of H3 and H4 and one heterodimer consisting of H2A and H2B. Thus, free histones are referred to herein and may be monomeric, heterodimeric, or tetrameric histones that do not assemble in a ("stoichiometric") macromolecular complex, such as nucleosomes, composed of histone octamers bound to nucleic acids. In addition, free histones can also bind to nucleic acids, and wherein the free histones are not assembled in ("stoichiometric") macromolecular complexes, such as intact nucleosomes. Preferably, the one or more free histones are substantially free of nucleic acids.

Lactate or lactic acid having the formula CH ₃ Organic compounds of CH (OH) COOH, which occur in body fluids comprising blood. Lactate was subjected to a blood test to determine the state of acid-base balance in the body. Lactic acid is a cellular metabolite that accumulates when cells lack sufficient oxygen (hypoxia) and must be diverted to less efficient energy production means, or when conditions lead to overproduction of lactate or impaired clearance. Lactic acidosis can be caused by an insufficient amount of oxygen (hypoxia) in cells and tissues, for example, if a person suffers from a condition that can result in a decrease in the amount of oxygen delivered to cells and tissues (such as shock, septic shock, or congestive heart failure), lactate testing can be used to help detect and assess the severity of hypoxia and lactic acidosis.

C-reactive protein (CRP) is a pentameric protein, which can be found in body fluids such as plasma. CRP levels can increase in response to inflammation. Measuring and plotting CRP values can prove useful in determining disease progression or the effectiveness of treatment.

As used herein, a "sequential organ failure assessment score" or "SOFA score" is one score used to track the status of a patient during stay in an Intensive Care Unit (ICU). SOFA score is a scoring system used to determine the extent of a person's organ function or failure rate. The scores are based on six different scores, each for the respiratory system, cardiovascular system, liver system, coagulation system, renal system and nervous system. Both the average SOFA score and the highest SOFA score are efficacy predictors. The increase in SOFA score during the first 24 to 48 hours in the ICU predicts mortality of at least 50% and as high as 95%. Scores below 9 give a predicted mortality rate of 33%, whereas scores above 14 approach or are above 95%.

As used herein, rapid SOFA score (qSOFA) is a scoring system that indicates a patient's risk of organ dysfunction or mortality. The scoring is based on three criteria: 1) change of mental state, 2) decrease of systolic blood pressure to less than 100mm Hg, 3) respiration rate greater than 22 breaths per minute. Patients with two or more of these conditions are at greater risk of organ dysfunction or death.

As used herein, "APACHE II" or "acute physiological and chronic health assessment II" is a disease severity classification scoring system (Knaus et al, 1985). It can be applied within 24 hours of taking the patient to an Intensive Care Unit (ICU) and can be determined based on 12 different physiological parameters: aaDO2 or PaO2 (depending on FiO 2), temperature (rectal), mean arterial pressure, arterial pH, heart rate, respiratory rate, sodium (serum), potassium (serum), creatinine, hematocrit, white blood cell count, and glasgang coma index.

As used herein, "SAPS II" or "simplified acute physiological score II" refers to a system for classifying the severity of a disease or disorder (see Le Gall JR et al, new simplified acute physiological score (SAPS II) based on European/North American Multi-center studies (A new Simplified Acute Physiology Score (SAPS II) based on a European/North American multicenter study) & JAMA 1993;270 (24) & 2957-63). The SAPS II score consisted of 12 physiological variables and 3 disease-related variables. Scores were calculated from 12 conventional physiological measurements, information about previous health conditions, and some information obtained when the ICU was entered. The SAPS II score may be determined at any time, preferably on day 2. The "worst" measurement is defined as the measure associated with the highest score. SAPS II scores ranged from 0 to 163 points. The classification system comprises the following parameters: age, heart rate, systolic pressure, temperature, glasgow coma index, mechanical ventilation or CPAP, paO2, fiO2, urine output, blood urea nitrogen, sodium, potassium, bicarbonate, bilirubin, leukocytes, chronic diseases and hospitalization type. There is an sigmoid relationship between mortality and SAPS II total score. The mortality of the subjects was 10% at a sapii score of 29, 25% at a sapii score of 40, 50% at a sapii score of 52, 75% at a sapii score of 64, and 90% at a sapii score of 77 (Le gal, cited above).

As used herein, the term "sample" is a biological sample obtained or isolated from a patient or subject. As used herein, a "sample" may refer, for example, to a sample of bodily fluid or tissue obtained for the purpose of diagnosis, prognosis, or evaluation of a subject of interest (e.g., a patient). Preferably herein, the sample is a sample of a bodily fluid, such as blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, pleural effusion, cells, cell extracts, tissue samples, tissue biopsies, stool samples, etc. Specifically, the sample is blood, plasma, serum or urine.

Embodiments of the present invention relate to the separation of a first sample and the separation of a second sample. In the context of the method of the present invention, the terms "first sample" and "second sample" relate to a relative determination of the separation order of the samples employed in the method of the present invention. When the terms first and second sample are used to describe the present method in detail, these samples should not be taken as an absolute determination of the number of samples taken. Thus, additional samples may be isolated from the patient before, during or after the isolation of the first sample and/or the second sample, or between the first sample or the second sample, wherein these additional samples may or may not be used in the methods of the invention. Thus, the first sample may be considered to be any previously obtained sample. The second sample may be considered any additional or subsequent sample.

In the context of the present invention, "plasma" is the almost cell-free blood supernatant containing anticoagulant obtained after centrifugation. Exemplary anticoagulants include calcium ion binding compounds (e.g., EDTA or citrate) and thrombin inhibitors (e.g., heparin salts or hirudin). Cell-free plasma may be obtained by centrifuging anticoagulated blood (e.g., citrate blood, EDTA blood, or heparinized blood), e.g., at 2000 to 3000g, for at least 15 minutes.

In the context of the present invention, "serum" is the liquid portion of whole blood collected after allowing the blood to coagulate. When the coagulated blood (coagulated blood) is centrifuged, serum can be obtained as a supernatant.

As used herein, "urine" is the fluid product of the body secreted by the kidneys through a process known as urination (or urination) and excreted through the urethra.

In a preferred embodiment of the invention, the patient has been diagnosed with sepsis. More specifically, the patient may have been diagnosed with severe sepsis and/or septic shock.

In the context of the present invention, "sepsis" refers to a systemic reaction to an infection. Alternatively, sepsis may be considered as a combination of SIRS with a confirmed course of infection or infection. Sepsis may be characterized as a clinical syndrome defined by the presence of both infection and systemic inflammatory response (Levy MM et al, 2001 SCCM/ESICM/ACCP/ATS/SIS International sepsis definition conference (SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference) & Critical Care Med.) & 2003, month 4; 31 (4) & 1250-6). The term "sepsis" as used herein includes, but is not limited to, sepsis, severe sepsis, septic shock.

The term "sepsis" as used herein includes, but is not limited to, sepsis, severe sepsis, septic shock. Severe sepsis refers to sepsis associated with organ dysfunction, hypoperfusion abnormalities, or sepsis-induced hypotension. Hypoperfusion abnormalities include lactic acidosis, oliguria, and acute changes in mental state. Sepsis-induced hypotension is defined by the presence of systolic blood pressure below about 90mm Hg or a reduction of about 40mm Hg or more from baseline in the absence of other causes of hypotension (e.g., cardiogenic shock). Septic shock is defined as severe sepsis in which sepsis-induced hypotension persists despite adequate fluid resuscitation with the concomitant presence of hypoperfusion abnormalities or organ dysfunction (Bone et al, thoracic (CHEST) 101 (6): 1644-55, 1992).

Alternatively, the term sepsis may also be defined as a life threatening organ dysfunction caused by a deregulation of the host's response to an infection. For clinical manipulation, organ dysfunction may be preferentially indicated by a 2 point or more elevation in Sequential Organ Failure Assessment (SOFA) score, which is associated with greater than 10% hospitalization mortality. Septic shock can be defined as a subgroup of sepsis in which particularly severe circulatory, cellular and metabolic abnormalities are associated with a greater risk of mortality than sepsis alone. Clinically, patients suffering from septic shock can be identified by maintaining an average arterial pressure of 65mm Hg or greater and a serum lactate level of greater than 2mmol/L (> 18 mg/dL) in the absence of hypovolemia.

The term "sepsis" as used herein relates to all possible stages in the development of sepsis.

The term "SEPSIS" also encompasses severe SEPSIS or septic shock based on the definition of SEPSIS-2 (SEPSIS-2) (Bone et al 2009). The term "SEPSIS" also encompasses subjects falling within the definition of SEPSIS-3 (SEPSIS-3) (Singer et al 2016). The term "sepsis" as used herein relates to all possible stages in the development of sepsis.

As used herein, "infection" within the scope of the present invention means a pathological process caused by invasion of normal sterile tissue or fluid by a pathogenic or potentially pathogenic agent/pathogen, organism and/or microorganism and preferably involves one or more infections by bacteria, viruses, fungi and/or parasites. Thus, the infection may be a bacterial infection, a viral infection, and/or a fungal infection. The infection may be a local or systemic infection. For the purposes of the present invention, a viral infection may be considered to be an infection by a microorganism.

Further, a subject with an infection may have more than one source of infection at the same time. For example, a subject with an infection may have a bacterial infection and a viral infection; with viral and fungal infections; with bacterial and fungal infections, as well as with bacterial, fungal and viral infections, or with mixed infections, including one or more of the infections listed herein, potentially including repeated infections, such as one or more bacterial infections in addition to one or more viral and/or one or more fungal infections.

As used herein, "infectious disease" includes all diseases or conditions associated with bacterial and/or viral and/or fungal infections.

Critical patients, such as sepsis patients, may require strict control of vital functions and/or monitoring of organ protection and may be undergoing medical treatment according to the present invention.

In the context of the present invention, the term "medical treatment" or "treatment" includes various treatment and therapeutic strategies including, but not limited to, anti-inflammatory strategies, administration of proADM antagonists such as therapeutic antibodies si-RNA or DNA, in vitro blood purification or removal of harmful substances by apheresis, dialysis, absorber to prevent cytokine storm, removal of inflammatory mediators, plasmapheresis, administration of vitamins such as vitamin C, ventilation such as mechanical ventilation and non-mechanical ventilation to provide sufficient oxygen to the body, focal clearance, blood product perfusion, colloid infusion, kidney or liver replacement, antibiotic therapy, invasive mechanical ventilation, non-invasive mechanical ventilation, kidney replacement therapy, use of vasopressors, fluid therapy, isolation and organ protection measures, provision of corticosteroids, transfusion or platelet infusion, infusion of blood components (such as serum, plasma or specific cells or combinations thereof), medicaments to promote platelet formation, treatment or splenectomy.

Additional treatments of the invention include administration of cells or cell products (such as stem cells, blood or plasma) and stabilization of the patient's circulation and protection of endothelial glycocalyx, e.g., by optimal fluid management strategies, e.g., to achieve normal blood volume and prevent or treat hypervolemia or hypovolemia. Furthermore, vasopressors or heparinase inhibition such as catecholamines and albumin or re-N-acetylated heparin by unfractionated heparin or N-desulphation are useful treatments to support circulation and endothelial layers.

In addition, medical treatments of the present invention include, but are not limited to, stabilization of blood coagulation, iNOS inhibitors, anti-inflammatory agents such as hydrocortisone, sedatives and analgesics, and insulin.

"renal replacement therapy" (RRT) refers to therapy for replacing the normal hemofiltration function of the kidneys. Renal replacement therapy may refer to dialysis (e.g., hemodialysis or peritoneal dialysis), hemofiltration, and hemodiafiltration. Such techniques are various methods of transferring blood to a machine, cleaning the blood, and then returning the blood to the body. Renal replacement therapy may also be referred to as kidney transplantation, which is the final alternative, as the old kidney is replaced by the donor kidney. Hemodialysis, hemofiltration, and hemodiafiltration may be continuous or intermittent and may use an arteriovenous route (where blood leaves from the artery and returns through the vein) or a venous route (where blood leaves from the vein and returns through the vein). This produces various types of RRT. For example, the renal replacement therapy may be selected from, but is not limited to, the group of: continuous Renal Replacement Therapy (CRRT), continuous Hemodialysis (CHD), continuous arteriovenous hemodialysis (CAVHD), continuous venous hemodialysis (CVVHD), continuous Hemofiltration (CHF), continuous arteriovenous hemofiltration (CAVH or CAVHF), continuous venous hemofiltration (CVVH or CVVHF), continuous Hemodiafiltration (CHDF), continuous arteriovenous hemodiafiltration (CAVHDF), continuous venous hemodiafiltration (CVVHDF), intermittent Renal Replacement Therapy (IRRT), intermittent Hemodialysis (IHD), intermittent venous hemodialysis (IVVHD), intermittent Hemofiltration (IHF), intermittent venous hemofiltration (IVVH or IVVHF), intermittent Hemodiafiltration (IHDF), and intermittent venous hemodiafiltration (IVVHDF).

Artificial ventilation and mechanical ventilation are effective methods for enhancing proper gas exchange and ventilation and aim to save lives during severe hypoxia. Artificial ventilation involves assisting or stimulating the respiration of a subject. Artificial ventilation may be selected from the group consisting of mechanical ventilation, manual ventilation, external membrane oxygenation (ECMO), and non-invasive ventilation (NIV). Mechanical ventilation relates to a method for mechanically assisting or replacing spontaneous breathing. This may involve a machine known as a ventilator. The mechanical ventilation may be high frequency oscillation ventilation or partial liquid ventilation.

"fluid management" refers to monitoring and controlling a fluid condition of a subject and administering the fluid to stabilize circulation or organ viability by, for example, oral, enteral, or intravenous fluid administration. Fluid management includes stabilizing fluid and electrolyte balance or preventing or correcting hypervolemia or hypovolemia and supplying blood products.

Surgical emergency/emergency surgery is required if the subject has a medical emergency and requires immediate surgical treatment to maintain survival or health. The subject in need of emergency surgery may be selected from the group consisting of patients suffering from acute trauma, active uncontrolled infection, organ transplantation, organ preventative or organ stable surgery or cancer.

The cleaning procedure is a hygienic method for preventing infections, in particular nosocomial infections, in a subject, comprising disinfecting all organic and inorganic surfaces that may come into contact with the patient, such as for example skin, objects in the patient's room, medical devices, diagnostic devices or room air. The cleaning procedure involves the use of protective clothing and units (such as mouth guards, gowns, gloves, or sanitary locks) and limited patient visits. In addition, the cleaning procedure includes cleaning the patient himself and clothing or the patient.

In the case of major diseases such as sepsis or severe infection, it is very important to diagnose the patient early and to prognosis and risk evaluate the patient's efficacy for optimal therapy and management. Therapeutic methods need to be very unique and situation-specific. Therapeutic monitoring is required for best practice therapy and is affected by the timing of treatment, use of combination therapy and optimization of drug administration. Incorrect or omitted therapy or management will increase mortality hourly.

The medical treatment of the present invention may be an antibiotic treatment, wherein one or more "antibiotics" or "antibiotic agents" may be administered if an infection has been diagnosed or symptoms of an infectious disease have been determined.

In addition, antibiotics include bacteriophages, synthetic antibacterial peptides or iron antagonists/iron chelators for the treatment of bacterial infections. In addition, therapeutic antibodies or antagonists administered against pathogenic structures (e.g., anti-VAP-antibodies), resistant clone vaccination, immune cells (e.g., T effector cells sensitized or modulated in vitro) are antibiotic agents that represent treatment options for critically ill patients such as sepsis patients. Additional antibiotic agents/treatment or therapeutic strategies for infections or for preventing new infections include the use of preservatives, decontamination products, antitoxic agents such as liposomes, environmental sanitation, wound care, surgery. Several of the above-described antibiotic agents or therapeutic strategies may also be combined with fluid therapy, platelet infusion, or blood product infusion.

According to the invention proADM and optionally PCT and/or other markers or clinical scores are employed as markers for therapy monitoring including prognosis, risk assessment and risk stratification of subsequent adverse events in the health of patients diagnosed as critically ill.

The skilled person is able to obtain or develop means for identifying, measuring, determining and/or quantifying the aforementioned proADM molecules or fragments or variants thereof, as well as other markers of the invention, according to standard molecular biological practices.

The level of proADM or a fragment thereof, as well as the level of other markers of the invention, can be determined by an assay that reliably determines the concentration of the marker. In particular, mass Spectrometry (MS) and/or immunoassays may be employed, as exemplified in the accompanying examples. As used herein, an immunoassay is a biochemical test that measures the presence or concentration of macromolecules/polypeptides in solution by using antibodies or antibody binding fragments or immunoglobulins.

Methods of determining proADM or other markers (e.g., PCT) used in the context of the present invention are contemplated in the present invention. By way of example, a method selected from the group consisting of: mass Spectrometry (MS), luminescent Immunoassay (LIA), radioimmunoassay (RIA), chemiluminescent immunoassay and fluorescent immunoassay, enzyme Immunoassay (EIA), enzyme Linked Immunoassay (ELISA), luminescence-based bead array, magnetic bead-based array, protein microarray assay, rapid test format (such as, for example, immunochromatographic strip test), rare hole complex assay and automated system/analyzer.

Determination of proADM and optionally other markers based on antibody recognition is a preferred embodiment of the invention. As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds to (immunoreacts with) an antigen. According to the invention, the antibody may be a monoclonal antibody or a polyclonal antibody. In particular, antibodies are used which specifically bind to at least proADM or a fragment thereof.

An antibody is considered specific if its affinity for a molecule of interest (e.g. proADM) or a fragment thereof is at least 50-fold, preferably 100-fold, most preferably at least 1000-fold higher than for other molecules comprised in a sample containing the molecule of interest. How to develop and select antibodies with a given specificity is well known in the art. In the context of the present invention, monoclonal antibodies are preferred. The antibody or antibody binding fragment specifically binds to a marker as defined herein or a fragment thereof. In particular, the antibody or antibody binding fragment binds to a proADM peptide as defined herein. Thus, a peptide as defined herein may also be an epitope to which an antibody specifically binds. Further, antibodies or antibody binding fragments that specifically bind to proADM or proADM, in particular MR-proADM, are used in the methods and kits of the invention.

Further, antibodies or antibody binding fragments that specifically bind to proADM or fragments thereof and optionally other markers of the invention (e.g., PCT) are used in the methods and kits of the invention. Exemplary immunoassays may be light emitting immunoassays (LIA), radioimmunoassays (RIA), chemiluminescent immunoassays and fluorescent immunoassays, enzyme Immunoassays (EIA), enzyme Linked Immunoassays (ELISA), light emitting bead arrays, magnetic bead based arrays, protein microarray assays, rapid test formats, rare hole compound assays. Further, assays suitable for both point-of-care and rapid testing formats (such as, for example, immunochromatographic strip tests) can also be employed. Automated immunoassays are also contemplated, such as the KRYPTOR assay.

Alternatively, the scope of the invention may encompass other capture molecules or molecular backbones that specifically and/or selectively recognize proADM instead of antibodies. In this context, the term "capture molecule" or "molecular scaffold" includes molecules that can be used to bind to a target molecule or a molecule of interest, i.e. an analyte (e.g. proADM, MR-proADM and PCT) from a sample. Thus, the capture molecules must be sufficiently shaped both spatially and in terms of surface features such as surface charge, hydrophobicity, hydrophilicity, presence or absence of Lewis donors and/or acceptors to specifically bind the target molecule or molecules of interest. Thus, binding may be mediated, for example, by: ion interactions, van der Waals interactions, pi-pi interactions, sigma-pi interactions, hydrophobic interactions or hydrogen bonding interactions or a combination of two or more of the foregoing interactions or covalent interactions between a capture molecule or molecular scaffold and a target molecule or molecule of interest. In the context of the present invention, the capture molecule or molecular scaffold may for example be selected from the group consisting of nucleic acid molecules, carbohydrate molecules, PNA molecules, proteins, peptides and glycoproteins. The capture molecule or molecular scaffold comprises for example an aptamer, DARpin (engineered ankyrin repeat protein). Including Affimer, etc.

In certain aspects of the invention, the method is an immunoassay comprising the steps of:

a) The sample was contacted with:

i. a first antibody or antigen binding fragment or derivative thereof specific for a first epitope of said proADM, and

a second antibody or antigen binding fragment or derivative thereof specific for a second epitope of said proADM; and

b) Detecting binding of said two antibodies or antigen binding fragments or derivatives thereof to said proADM.

Preferably, one of the antibodies may be labeled and the other antibody may be bound to a solid phase or may be selectively bound to a solid phase. In a particularly preferred aspect of the assay, one of the antibodies is labelled and the other antibody is bound to a solid phase or may be selectively bound to a solid phase. The first antibody and the second antibody may be present in a dispersed form in a liquid reaction mixture, and wherein a first label component, which is part of a fluorescence-based or chemiluminescent extinction or amplification-based label system, is bound to the first antibody and a second label component of the label system is bound to the second antibody, such that upon detection of binding of the two antibodies to the proADM or fragment thereof, a measurable signal is generated that allows detection of the resulting sandwich complex in a measurement solution. The labelling system may comprise rare earth cryptates or chelates in combination with fluorescent or chemiluminescent dyes, in particular cyanine-type dyes.

In a preferred embodiment, the method is performed as a heterogeneous sandwich immunoassay, wherein one of the antibodies is immobilized on an arbitrarily selected solid phase, e.g. a coated tube (e.g. a polystyrene tube; a coated tube; CT) or a wall of a microtiter plate (e.g. composed of polystyrene), or to particles, such as e.g. magnetic particles, whereby the other antibody has a group similar to a detectable label or effecting selective attachment to a label, and the group is used for detecting the formed sandwich structure. It is also possible to delay the immobilization temporarily or to carry out subsequent immobilization by using a suitable solid phase.

The method according to the invention may further be embodied as a homogeneous method, wherein the sandwich complex formed by one/both antibodies and the marker to be detected, i.e. proADM or a fragment thereof, is kept suspended in a liquid phase. In this case, it is preferred that when two antibodies are used, they are labeled with parts of the detection system, which results in the generation of a signal or triggering of a signal when the two antibodies are integrated into a single sandwich structure. Such techniques should be embodied as fluorescence enhancement or fluorescence quenching detection methods. Particularly preferred aspects relate to that should be paired The use of detection reagents used, such as for example those described in US4882733, EP0180492 or EP0539477 and the prior art cited therein. In this way, it becomes possible to detect only the measurement of the reaction products directly comprising the two marker components in a single immunocomplex in the reaction mixture. For example, such techniques are under the trade name(time resolved amplification of hole complex emission) orIs provided to enable the teaching of the above application. Thus, in a particularly preferred aspect, the methods provided herein are performed using a diagnostic device. For example, the level of proADM or a fragment thereof and/or the level of any additional marker (e.g., PCT) of the methods provided herein is determined. In a particularly preferred aspect, the diagnostic device is +.>

The level of a marker of the invention, e.g. proADM or a fragment thereof, PCT or a fragment thereof or other marker, can also be determined by means of a Mass Spectrometry (MS) based method. Such methods may comprise detecting the presence, amount or concentration of one or more modified or unmodified fragment peptides, e.g. proADM or PCT, in the biological sample or protein digests (e.g. trypsin digests) from the sample, and optionally separating the sample by chromatography, and performing MS analysis on the prepared and optionally separated sample. For example, selective Reaction Monitoring (SRM), multiple Reaction Monitoring (MRM) or Parallel Reaction Monitoring (PRM) mass spectrometry may be used for MS analysis, in particular in order to determine the amount of proADM or fragments thereof.

Herein, the term "mass spectrometry" or "MS" refers to an analytical technique for identifying a compound by its mass. In order to enhance mass resolving and mass determining capabilities of mass spectrometry, samples may be processed prior to MS analysis. The present invention thus relates to a method of MS detection that can be associated with immune enrichment techniques, and sample preparation and/or chromatography, preferably with Liquid Chromatography (LC), more preferably with High Performance Liquid Chromatography (HPLC) or Ultra High Performance Liquid Chromatography (UHPLC). Sample preparation methods include techniques for lysing, fractionating, digesting the sample into peptides, depleting, enriching, dialyzing, desalting, alkylating and/or reducing the peptides. However, these steps are optional. Selective detection of analyte ions can be performed using tandem mass spectrometry (MS/MS). Tandem mass spectrometry is characterized by a mass selection step (as used herein, the term "mass selection" means the separation of ions having a defined m/z or narrow m/z range), followed by fragmentation of the selected ions and mass analysis of the resulting product (fragment) ions.

The skilled person realizes how to quantify the level of a marker in a sample by mass spectrometry. For example, as described above, a relative quantification "rSRM" or absolute quantification may be employed.

In addition, the level (including the reference level) may be determined by mass spectrometry-based methods, such as methods of determining the relative quantification of the protein of interest or fragment thereof or determining the absolute quantification of the protein of interest or fragment thereof.

The relative quantification "rSRM" can be achieved by:

1. the increased or decreased presence of a target protein is determined by comparing the SRM (selective reaction monitoring) characteristic peak area of a given target fragment peptide from detection in a sample with the same SRM characteristic peak area of the target fragment peptide in at least a second, third, fourth or more biological samples.

2. The increased or decreased presence of a target protein is determined by comparing the SRM characteristic peak area from a given target peptide detected in a sample with SRM characteristic peak areas generated from fragment peptides of other proteins in other samples derived from different individual biological sources, wherein the SRM characteristic peak area comparison between the two samples of peptide fragments is normalized with respect to, for example, the amount of protein analyzed in each sample.

3. The increased or decreased presence of a target protein is determined by comparing the SRM characteristic peak area of a given target peptide with the SRM characteristic peak areas of other fragment peptides derived from different proteins within the same biological sample in order to normalize the level of change in histone protein relative to the level of other proteins whose expression level is not changed under various cellular conditions.

4. These assays can be applied to both unmodified and modified fragment peptides of a target protein, wherein the modification includes, but is not limited to, phosphorylation and/or glycosylation, acetylation, methylation (monomethylation, dimethylation, trimethylation), citrullination, ubiquitination, and wherein the relative levels of modified peptides are determined in the same manner as the relative amounts of unmodified peptides are determined.

Absolute quantification of a given peptide can be achieved by:

1. the SRM/MRM characteristic peak areas of a given fragment peptide from a target protein in a biological sample alone are compared to the SRM/MRM characteristic peak areas of an internal fragment peptide standard incorporated into a protein lysate from the biological sample. The internal standard may be a labeled synthetic version of a fragment peptide from the target protein or labeled recombinant protein being interrogated. This standard is incorporated into the sample either before digestion (mandatory for recombinant proteins) or after digestion in known amounts, and the SRM/MRM characteristic peak areas of both the internal fragment peptide standard and the native fragment peptide in the biological sample can be determined separately, and then the two peak areas compared. This may apply to unmodified fragment peptides and modified fragment peptides, wherein the modification includes, but is not limited to, phosphorylation and/or glycosylation, acetylation, methylation (e.g., monomethylation, dimethylation, trimethylation), citrullination, ubiquitination, and wherein the absolute level of modified peptide is determined in the same manner as the absolute level of unmodified peptide is determined.

2. The peptides can also be quantified using an external calibration curve. The normal curve method uses constant amounts of heavy peptide as an internal standard and different amounts of light synthetic peptide incorporated into the sample. A standard curve needs to be constructed using a representative matrix similar to the test sample to account for matrix effects. In addition, the inverse curve approach circumvents the problem of endogenous analytes in a matrix, where a constant amount of light peptide is incorporated on top of the endogenous analyte to produce an internal standard and different amounts of heavy peptide are incorporated to produce a set of concentrated standards. The test sample to be compared to the normal or reverse curve is doped with the same amount of standard peptide as the internal standard incorporated into the matrix used to generate the calibration curve.

The invention further relates to kits, uses of the kits and methods in which such kits are used. The present invention relates to kits for performing the methods provided herein above and below. The definitions provided herein, for example in relation to the methods, also apply to the kits of the invention. In particular, the invention relates to a kit for therapy monitoring comprising prognosis, risk assessment or risk stratification for subsequent adverse events in the health of a patient, wherein the kit comprises:

-a detection reagent for determining the level of proADM or one or more fragments thereof in a sample from a subject and optionally additionally for determining the level of PCT, lactate and/or C-reactive protein or one or more fragments thereof, and a detection reagent for determining said level of proADM in said sample of said subject, and

-reference data corresponding to a high and/or low severity level of proADM, such as a reference level, wherein the low severity level is below 4nmol/l, preferably below 3nmol/l, more preferably below 2.7nmol/l, and the high severity level is above 6.5nmol/l, preferably above 6.95nmol/l, more preferably above 10.9nmol/l, and optionally PCT, lactate and/or C-reactive protein levels, wherein the reference data is preferably stored on a computer readable medium and/or used in the form of computer executable code configured to compare the determined level of proADM or one or more fragments thereof and optionally the determined level of further PCT, lactate and/or C-reactive protein or one or more fragments thereof with the reference data.

As used herein, "reference data" includes one or more reference levels of proADM and optionally PCT, lactate and/or C-reactive protein. Levels of proADM and optionally PCT, lactate and/or C-reactive protein in a sample of a subject can be compared to reference levels included in reference data of the kit. Reference levels are described above and are also exemplified in the examples that follow. The reference data may also comprise a reference sample to which the levels of proADM and optionally PCT, lactate and/or C-reactive protein are compared. The reference data may also contain instruction manuals of how to use the kits of the invention.

The kit may additionally comprise an article useful for obtaining a sample such as a blood sample (e.g., the kit may comprise a container, wherein the container comprises means for attaching the container to a cannula or syringe), a syringe adapted for blood separation, an internal pressure exhibiting a pressure below atmospheric pressure (e.g., adapted for drawing a predetermined volume of sample into the container), and/or additionally comprise a detergent, a chaotropic salt, a ribonuclease inhibitor, a chelating agent (e.g., guanidinium isothiocyanate, guanidinium hydrochloride, sodium dodecyl sulfate, polyoxyethylene sorbitan monolaurate, RNAse inhibitor protein, and mixtures thereof), and/or a filtration system comprising: nitrocellulose, silica matrix, ferromagnetic spheres, recycled spill cups (a cup retrieve spill over), trehalose, fructose, lactose, mannose, polyethylene glycol, glycerol, EDTA, TRIS, limonene, xylenes, benzoyl, phenol, mineral oil, aniline, pyrrole, citrate and mixtures thereof.

As used herein, "detection reagent" and the like are reagents suitable for determining markers for e.g. proADM, PCT, lactate and/or C-reactive proteins described herein. Such exemplary detection reagents are, for example, ligands, such as antibodies or fragments thereof, that specifically bind to peptides or epitopes of one or more of the markers described herein. Such ligands may be used in immunoassays as described above. Additional reagents for determining the level of the one or more markers in an immunoassay may also be included in the kit and are considered herein as detection reagents. The detection reagent may also relate to a reagent for detecting a marker or a fragment thereof by an MS-based method. Thus, such detection reagents may also be reagents for preparing samples for MS analysis, such as enzymes, chemicals, buffers, etc. Mass spectrometers can also be regarded as detection reagents. The detection reagent according to the invention may also be one or more calibration solutions, which may be used, for example, to determine and compare the level of the one or more markers.

The sensitivity and specificity of a diagnostic and/or prognostic test is not only dependent on the analytical "quality" of the test, but also on the definition of the features that make up the abnormal result. In practice, the subject's working characteristics (ROC curves) are typically calculated by plotting the values of the variables of the "normal" population (i.e., apparently healthy individuals not suffering from infection) and the "diseased" population (e.g., subjects suffering from infection) against their relative frequencies. For any particular marker (e.g., proADM), the distribution of marker levels in subjects with and without disease/condition may overlap. Under such conditions, the test does not absolutely distinguish between normal and disease with 100% accuracy, and the overlapping region may indicate that the test is unable to distinguish between normal and disease conditions. A threshold is selected below which a test is considered abnormal, and above which a test is considered normal, or below or above which a test indicates a particular condition, such as an infection. The area under the ROC curve is a measure of the likelihood that a perceived measurement will allow for the correct identification of a pathology. Even if the test results do not necessarily give exact numbers, ROC curves can be used. As long as the results can be ordered, ROC curves can be generated. For example, the test results of "disease" samples may be ranked according to extent (e.g., 1=low, 2=normal, and 3=high). This ordering can be correlated with results in the "normal" population, and a ROC curve is generated. Such methods are well known in the art; see, e.g., hanley et al, 1982 (Radiology) 143:29-36. Preferably, the threshold is selected to provide a ROC curve area greater than about 0.5, more preferably greater than about 0.7, still more preferably greater than about 0.8, even more preferably greater than about 0.85, and most preferably greater than about 0.9. In this context, the term "about" refers to +/-5% of a given measurement.

The horizontal axis of the ROC curve represents (1-specificity), which increases with false positive rate. The vertical axis of the curve represents sensitivity, which increases with true positive rate. Thus, for a particular critical value selected, a value of (1-specificity) can be determined, and a corresponding sensitivity can be obtained. The area under the ROC curve is a measure of the likelihood that the measured marker level will allow for the correct identification of the disease or condition. Thus, the area under the ROC curve can be used to determine the validity of the test.

Thus, the invention includes administration of antibiotics suitable for treatment based on the information obtained by the methods described herein.

As used herein, the terms "comprises" and "comprising," or grammatical variants thereof, are to be taken as specifying the stated features, integers, steps or components, but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof. This term encompasses the terms "consisting of … …" and "consisting essentially of … …".

Thus, the term "comprising"/"including"/"having" means that any additional component (or, as such, feature, integer, step, etc.) may be present (can/can). The term "consisting of … …" means that any additional component (or, as such, feature, integer, step, etc.) may be present.

As used herein, the term "consisting essentially of … …" or grammatical variants thereof shall be taken to specify the stated features, integers, steps or components but does not preclude the addition of one or more additional features, integers, steps, components or groups thereof but only if the additional features, integers, steps, components or groups thereof do not substantially alter the basic and novel characteristics of the claimed composition, apparatus or method.

Thus, the term "consisting essentially of … …" means that those specific additional components (or, as such, features, integers, steps, etc.) may be present, i.e., those that do not substantially affect the essential characteristics of the composition, apparatus or method. In other words, the term "consisting essentially of … …" (which may be used interchangeably herein with "consisting essentially of") allows for the presence of other components in the composition, device or method in addition to the mandatory components (or likewise, features, integers, steps, etc.), provided that the presence of the other components does not substantially affect the essential characteristics of the device or method.

The term "method" refers to means, techniques, and procedures for accomplishing a given task including, but not limited to, those known to, or readily developed by, practitioners in the chemical, biological, and biophysical arts.

Drawings

Embodiments of the present invention are illustrated by the accompanying drawings, as follows:

fig. 1: the effect of fluid administration during the first 24 hours according to MR-proADM kinetics. A moderate correlation (r=0.595) was achieved based on mortality within these MR-proADM severity groups and development of RRT.

Fig. 2: the effect of fluid administration during the first 24 hours according to MR-proADM kinetics. A strong correlation can be found between each MR-proADM severity group, mortality and fluid administration (R ² ＝0.931)。

Fig. 3: the effect of fluid application during the first 24 hours according to lactate kinetics. Lactate values are evaluated in relation to the amount of fluid administered to the patient. Lactate levels show a similar relationship to fluid volumes compared to proADM. The correlation was comparable to that of MR-proADM (R ² ＝0.965)。

Fig. 4: the effect of colloidal administration during the first 4 days according to MR-proADM kinetics. A high correlation was achieved based on RRT requirements within the MR-proADM severity group and the volume of fluid administered (r=0.894).

Fig. 5: the effect of colloidal administration during the first 4 days according to MR-proADM kinetics. Similar results as in fig. 4 were also achieved when the patient's weight was taken into account, resulting in intravenous fluid administration rates in ml/kg and R ² 9.49.

Fig. 6: the effect of colloidal administration during the first 4 days according to lactate kinetics. And also applied to the affected partThe lactate value was assessed in relation to the fluid quantity. Lactate levels show a similar relationship to fluid volumes compared to proADM. However, the correlation is significantly weaker than (R ² =0.810) correlation of MR-proADM.

The invention is further described by reference to the following non-limiting examples.

Examples

The method of the example:

study design and patient:

the study was a secondary analysis of placebo controlled trial (SISPCT) (26) of severe sepsis sodium selenite and procalcitonin-directed antibacterial therapy conducted across 33 multidisciplinary Intensive Care Units (ICU) across germany from 11 months 2009 to 2 months 2013. The qualifying criteria contained adult patients ≡18 years old who developed new severe sepsis or septic shock (+.24 hours) according to the definition of sepsis-1 by the ACCP/SCCM consensus conference committee (ACCP/SCCM Consensus Conference Committee), and were further classified according to the definition of 2016 (sepsis-3 and septic shock-3) (4). Details of study design, data collection and management have been previously described (26). The ethics committee of the university of jean hospital (Jena University Hospital) and all other centers approved the study and informed written consent was obtained when necessary.

Biomarker measurement:

patients were enrolled up to 24 hours after the diagnosis of severe sepsis or septic shock, and PCT, CRP and lactate were measured immediately thereafter. PCT was measured on a device with a measurement range of 0.02-5000ng/ml and a functional assay sensitivity and lower detection limit of at least 0.06ng/ml and 0.02ng/ml, respectively. Additional blood samples from all patients were collected and stored at-80 ℃ in a jean's central research laboratory. The MR-proADM plasma concentration was measured retrospectively with a detection limit of 0.05nmol/L (Sesameimeric Feishr science and technology, germany). At the time of study entry, the acquisition includes a sequenceOrgan Failure Assessment (SOFA) score, acute physiological and chronic health assessment (APACHE) II score, and clinical severity score that Simplifies Acute Physiological (SAPS) II score.

Statistical analysis:

differences in demographic and clinical characteristics with respect to 28-day mortality were assessed using a χ2 test for the class variable and a student t-test or Mann-Whitney U test (Mann-Whitney U test) for the continuous variable according to distribution normalization. The normal distribution variable and the non-normal distribution variable are expressed as an average value (standard deviation) and a median value [ first quartile-third quartile ], respectively. The correlation between mortality and each biomarker and clinical score at all time points was assessed using an area under the subject's working characteristics curve (AUROC) and Cox regression analysis, where the multivariate analysis was corrected for age and the presence of co-disease and septic shock. Patients were further divided into three severity subgroups (low, medium, high) based on calculation of two AUROC thresholds across the general population at each time point for each biomarker and clinical score, with predefined sensitivity and specificity approaching 90%. A clinically stable subset of patients were then identified as being devoid of any ICU-related surgery or complications (including focal clearance, emergency surgery, emerging new infections, blood product infusion, colloid infusion, invasive mechanical ventilation, renal/hepatic replacement or vasopressor medication, and exacerbation of the patient's general clinical signs and symptoms), while the other group was identified as having a corresponding low MR-proADM concentration that did not show any elevation since the previous measurement. Mortality and average hospitalization times for both groups were calculated and compared to the patient groups admitted to discharge at each specific time point.

Finally, two models were constructed that stratified the patient with 20% PCT change (baseline to day 1, based on the average PCT decrease observed during this period) and 50% PCT change (baseline to day 4, based on the previously constructed model (26)). Patient subgroups were then identified based on MR-proADM severity levels and corresponding mortality was calculated. Mortality risk within each subgroup was calculated by Cox regression analysis and demonstrated by Kaplan-Meier curves. The predicted risk of developing a new infection and the need for focal clearance and emergency procedures on days 4 to 7 was then studied in a baseline to day 4 model. All data were analyzed using statistical software R (version 3.1.2).

Example 1: patient characteristics

Patient characteristics at study entry are summarized in table 1.

A total of 1089 patients with severe sepsis (13.0%) or septic shock (87.0%) were analyzed, of which 445 (41.3%) and 633 (58.7%) also met the criteria for sepsis-3 and septic shock-3, respectively. The average age of the patients in the group was 65.7 (13.7) years and the average SOFA score was 10.0 (3.3) points. The 28-day total cause mortality (n=1076) was 26.9% (sepsis-3:20.0%; septic shock-3:32.1%), with hospitalized mortality 33.4% (sepsis-3:24.4%: septic shock-3:40.4%). Infection from a single lesion was found in 836 patients (77.7%), with lung disease sources (n=324; 30.1%), abdominal endogenous sources (n=252; 23.4%), genitourinary sources (n=57; 5.3%) and bone/soft tissue sources (n=50; 4.6%) being most common. The corresponding mortality rates were 26.5%, 24.6%, 22.8% and 28.0%, respectively. A variety of sources of infection were found in 240 (22.3%) patients. The most common causes of death include sepsis-induced multiple organ failure (n=132; 45.7%), refractory septic shock (n=54; 18.7%), death from existing diseases (n=35; 12.1%), and acute respiratory insufficiency (n=17; 5.9%). The combined mortality rate due to other causes such as cardiogenic and hemorrhagic shock, pulmonary embolism, cerebral edema, myocardial infarction and arrhythmia is 8.6%. The limitations of therapy apply to 3.4% of patients.

Example 2: correlation of baseline biomarkers and clinical scores with mortality

Univariate and multivariate Cox regression analysis found that MR-proADM correlated most strongly with 28-day mortality across the entire patient population and within the sepsis-3 and septic shock-3 subgroups (table 2). Corresponding AUROC analysis found significant differences in comparison to all biomarkers and clinical scores of MR-proADM, except APACHE II (subgroup of sepsis-3 patients).

Similar results were also found in 7 days, 90 days, ICU and hospitalized mortality predictions (table 3), where addition of MR-proADM to all potential biomarker and clinical score combinations (n=63) significantly improved prognostic power (table 4).

Example 3: identification of high risk patients

The entire patient population was further stratified according to the existing SOFA severity level and the performance of biomarkers and clinical scores in each subgroup was assessed at the time of predicting 28-day mortality. MR-proADM has the highest accuracy in all parameters in the low (SOFA.ltoreq.7) and moderate (8.ltoreq.SOFA.ltoreq.13) severity SOFA subgroups (Table 5; table 6).

Two corresponding MR-proADM thresholds were then calculated to identify low (. Ltoreq.2.7 nmol/L) and high (> 10.9 nmol/L) severity subgroups at baseline. More accurate reclassification can be performed at both low (MR-proADM vs SOFA: n=265 vs 232;9.8% vs 13.8% mortality) and high (MR-proADM vs SOFA: n=161 vs 155;55.9% vs 41.3%) severity thresholds compared to SOFA (table 7).

The 28-day mortality and 90-day mortality of the subgroup of 94 patients (9.3%) with high MR-proADM concentration and corresponding low or medium SOFA were 57.4% and 68.9%, respectively, compared to 19.8% and 30.8% in the remaining patient population with low and medium SOFA values. Similar patterns can be found in SAPS II, APACHE II and lactate, respectively (tables 8-10).

Example 4: identification of low risk patients throughout ICU hospitalization

The study cohort includes a subset of clinically stable patients who are not exposed to ICU-related surgery or complications such as focal clearance, emergency surgery, new-born infections, blood product infusion, colloid infusion, invasive mechanical ventilation, kidney/liver replacement, exacerbation of the patient's general clinical signs and symptoms.

This group of clinically stable patients is classified as low risk patients.

MR-proADM showed the strongest correlation with 28-day mortality across all subsequent time points (table 11), and could provide a stability threshold of ∈2.25nmol/L when identifying low risk patient populations, resulting in a larger patient number with lower mortality classified as compared to other biomarkers and clinical scores (table 12). Thus, 290 patients with low MR-proADM severity could be identified on day 4, of which 79 (27.2%) were clinically stable and had no increase in MR-proADM concentration compared to the previous measurement (Table 13). A sustained lower MR-proADM concentration was found in 51 (64.6%) patients, while a decrease from a moderate to a low severity level was observed in 28 (35.4%) patients. Average ICU hospitalization time was 8[7-10 days with 28-day mortality and 90-day mortality of 0.0% and 1.4%, respectively. In contrast, only 43 patients were actually admitted to leave the ICU on day 4, with 28-day mortality and 90-day mortality of 2.3% and 10.0%, respectively. Analysis of the MR-proADM concentrations for this group of patients indicated a range of values, of which 20 (52.6%), 16 (42.1%) and 2 (5.3%) patients had low, medium and high severity concentrations, respectively. Similar results were found in patients still in the ICU on days 7 and 10.

MR-proADM with a stability threshold of 2.25nmol/L can identify a larger number of low risk patients with lower mortality compared to other biomarkers and clinical scores. Based on the findings, more patients may be admitted to leave the ICU than would be classified without the ADM. By permitting more patients to be discharged, hospitals can use the ICU bed more efficiently and benefit from avoided costs.

Example 5: additional Effect of MR-proADM on procalcitonin-guided therapy

Time-dependent Cox regression analysis indicated that the earliest additional significant increase in prognostic information relative to the MR-proADM baseline could be observed on day 1, with subsequent single or cumulative measurements resulting in a significantly stronger association with 28-day mortality (table 14). Thus, two PCT guided algorithmic models were constructed to study PCT changes from baseline to day 1 or day 4, with corresponding subgroup analysis based on MR-proADM severity classification.

Patients with PCT concentrations decreasing by ≡20% from baseline to day 1 (table 15 and table 16) or by ≡50% from baseline to day 4 (table 17 and table 18) were found to have 28-day mortality of 18.3% (n=458) and 17.1% (n=557), respectively. This drops to 5.6% (n=125) and 1.8% (n=111) when the patient's MR-proADM level continues to be low, although rises to 66.7% (n=27) and 52.8% (n=39) (HR [95% ci ]:19.1[8.0-45.9] and 43.1[10.1-184.0 ]) in patients with MR-proADM values that continue to be high.

In addition, patients with a decrease in PCT value of > 50% (baseline to day 4) but with a continuously higher or moderate MR-proADM concentration are at significantly greater risk of developing subsequent nosocomial infections (HR [95% CI ]: high concentration: 3.9[1.5-10.5]; moderate concentration: 2.4[1.1-5.1] versus patient with a continuously lower concentration; moderate concentration: 2.9[1.2-6.8] versus moderate concentration down to low concentration), or require emergency surgery (HR [95% CI ]: moderate concentration: 2.0[1.1-3.7] versus moderate concentration down to low concentration). In contrast, patients with moderate to high concentrations are more likely to require removal of the infectious agent than patients with sustained moderate values (HR [95% CI ]:3.2[1.3-7.6 ]) or declining values (HR [95% CI ]: moderate to low: 8.7[3.1-24.8]; high to moderate: 4.6[1.4-14.5 ]). When PCT levels are not reduced by ≡50%, a significant increase in risk of requiring emergency surgery is observed if the MR-proADM concentration is at a continuously higher (HR [95% CI ]:5.7[1.5-21.9 ]) or medium (HR [95% CI ]:4.2[1.3-13.2 ]) level as opposed to a continuously lower one.

Example 6: correlation of baseline biomarkers and clinical scores with mortality

MR-proADM showed the strongest association in patients with pulmonary and intra-abdominal infections as well as in patients with gram-positive infections, regardless of the source of the infection (tables 19-20). MR-proADM provides the strongest and most balanced association with 28-day mortality across all groups when patients were grouped according to surgical emergency, non-surgical emergency and selective surgical history resulting in a live ICU (table 21).

Example 7: correlation of biomarkers and clinical scores with SOFA at baseline and day 1

MR-proADM had the greatest correlation between all biomarkers and the SOFA score at baseline, which increased significantly when the baseline value correlated with the 1 st-day SOFA score. The greatest correlation was found between MR-proADM and SOFA on day 10, with a difference between individual SOFA sub-scores always found (tables 22-24).

Example 8: identification of high risk patients

Similar results can be found in a subset of 124 (12.0%) patients with high MR-proADM concentration and low or moderate SAPS II values (high MR-proADM subset: [ mortality of 54.8% and 65.6% ], the remaining SAPS II population [ mortality of 19.7% and 30.0% ]) and in 109 (10.6%) patients with low or moderate APACHE II values (high MR-proADM subset: [ mortality of 56.9% and 66.7% ], the remaining APACHE II population: [ mortality of 19.5% and 30.3% ]).

Example 9: procalcitonin (PCT) -guided therapy improved by combining PCT with ADM

Two PCT guided algorithm models were constructed to study PCT changes from baseline to day 1 or day 4, with corresponding subgroup analysis based on MR-proADM severity classification (tables 25-30).

The previous examples show the additional value of ADM in patients with a decrease in PCT < 20% or < 50% and in patients with a decrease in PCT of > 20% or > 50%. However, additional analysis shows that ADM can be additional, regardless of the% decrease or even increase in PCT. The reduced PCT value may reflect the patient for whom the antibiotic treatment appears to be effective, so the clinician believes that the patient will survive soon (i.e., kill the underlying cause of sepsis-bacteria-should improve the patient).

For example, PCT levels for some patients decrease from baseline (day of admission) to day 1 with a mortality rate of 19% at 28 days. By additionally measuring ADM, it can be concluded that patients with low ADM have a higher chance of survival or a lower likelihood of death (Table 25; comparing only reduced PCT at mortality of 19% with PCT+low ADM at mortality of 5%). By reducing the risk of death, the patient may be more confident of getting away from the ICU, or require fewer diagnostic tests (i.e., be aware that the patient will recover soon).

On the other hand, new measures need to be considered for those patients with high ADM values. Their mortality risk is much higher (19% mortality in PCT reduced only compared to 58.8% mortality PCT + high ADM). The physician considers that the patient improved due to the decrease in PCT value, but in fact the ADM concentration remained unchanged. Thus, it can be concluded that the treatment is not active and needs to be adjusted as soon as possible.

In a similar manner, ADM may help to stratify those patients with increased PCT values (table 25).

Occurrence of new infection

PCT and MR-proADM changes from baseline to day 1 or from baseline to day 4 were analyzed in both models. Patients were grouped according to overall PCT changes and MR-proADM severity levels.

The new number of infections on day 1, day 2, day 3 and day 4 (table 26) and on day 4, day 5, day 6 and day 7 (table 27) were then calculated in each patient appearing on day 1 or day 4, respectively. In some cases, the patient is admitted for discharge during the observation period. It is assumed that no new infection occurs after discharge. Patients with multiple infections over the days of observation were considered as single new infections.

As a clinical result, it should be possible to treat patients with high MR-proADM concentrations with broad-spectrum antibiotics in combination with others after having entered the ICU to prevent new infections from occurring. These patients should be given special care since they are very susceptible to new infections.

Requirements for lesion clearance

The number of lesion clearance events was then calculated on day 1, day 2, day 3 and day 4 (table 28) and on day 4, day 5, day 6 and day 7 (table 29) in each patient appearing on day 1 or day 4, respectively. In some cases, the patient is admitted for discharge during the observation period.

Requirements for emergency operations

The emergency surgical needs/event count was then calculated on days 1, 2, 3 and 4 (table 30) for each patient present on day 1. In some cases, the patient is admitted for discharge during the observation period.

Example 10: requirements for antibiotic changes or modifications

MR-proADM, when combined in PCT-guided antibiotic algorithms, can stratify those patients who need to change or modify antibiotic therapy in the future from those patients who do not.

The percentage of antibiotic change on day 4 required for each patient group was then calculated (tables 31 and 32).

In patients with a decrease in PCT value of 50% or more

Patients with elevated MR-proADM concentrations from low to medium severity levels are more likely to require modification of antibiotic therapy on day 4 than those with consistently low levels (odds ratio [95% CI ]:1.5[0.6-4.1 ]).

In patients with a decrease in PCT value of < 50%

Patients with increasing concentrations of MR-proADM from moderate to high severity levels or with higher concentrations also have a higher likelihood of requiring changes in antibiotic therapy on day 4 than patients with lower concentrations of MR-proADM (5.9 [1.9-18.1] and 2.9[0.8-10.4] odds ratios [95% CI ]: respectively).

Conclusion(s)

Although PCT concentrations increase from baseline to day 1 or from baseline to day 4, patients with lower MR-proADM concentrations consistently have significantly less modifications to their prescribed antibiotic treatment than those with medium or higher concentrations. As a clinical result, the physician should examine the patient's MR-proADM levels in the face of elevated PCT concentrations before deciding to change antibiotics. Before considering the change, it should be considered to use an increased dose or an increased intensity of the same antibiotic for those patients with a low MR-proADM concentration. Early anti-biological changes (i.e. on days 1 to 3, as opposed to 4) should be considered for patients with higher MR-proADM concentrations.

Example 11: identification of patients with abnormal platelet levels and identification of high risk patients with thrombocytopenia (tables 33, 34 and 35).

The adrenomedullin precursor and procalcitonin levels were measured and analyzed with respect to platelet count, mortality and platelet infusion at baseline and day 1. Elevated proADM and PCT concentrations are associated with a reduced number of platelets and a number of platelets reflecting thrombocytopenia (< 150.000 per microliter). The most decrease in platelet count was observed in the patient with the highest proADM level at baseline. In addition, elevated proADM and PCT concentrations are consistent with patients in need of platelet infusion therapy. It could also be demonstrated that higher mortality is associated with thrombocytopenia and elevated levels of proADM (> 6 nmol/L) and PCT (> 7 ng/ml).

The pro-ADM levels were studied in patients with normal platelets at baseline to see if elevated levels of pro ADM could predict thrombocytopenia. Thrombocytopenia occurs at 39.4% of patients with continuously elevated levels of proADM (> 10.9 nmol/l) at baseline and on day 1. Thrombocytopenia occurs in 25.6% of patients with elevated levels of proADM at baseline (proADM > 2.75 nmol/L) and at day 1 (proADM > 9.5 nmol/L). Thrombocytopenia occurs in 14.7% of patients with consistently lower levels of proADM (proADM. Ltoreq.2.75 nmol/l) at baseline and on day 1. Elevated levels of proADM correlate with the severity of thrombocytopenia events and associated elevated mortality (proADM > 10.9nmol/L, mortality 51%; proADM. Ltoreq.2.75 nmol/L, mortality 9.1%). Example 11 relates to tables 33-35.

Example 12: effects of fluid volume on mortality and MR-proADM concentration

Emerging evidence suggests that the type and dosage of fluid therapy can affect patient outcome. The data set obtained in the secondary analysis of placebo-controlled test (SISPCT) (26) of severe sepsis sodium selenite and procalcitonin-directed antibacterial therapy described herein from 11 months 2009 to 2 months 2013 across 33 multidisciplinary Intensive Care Units (ICU) germany was evaluated by investigating the effect of fluid volumes on mortality and MR-proADM concentrations. There is currently no method of determining how much fluid or which fluid should be administered to a patient.

This analysis described in example 12 emphasizes the use of baseline, day 1 and day 4 MR-proADM concentrations in directing the volume of fluid to be administered in order to maintain low or reduce MR-proADM concentrations and direct the use of specific colloids according to MR-proADM concentrations.

The analysis presented herein shows that excessive administration of fluid volumes is often detrimental, resulting in organ dysfunction and progression to multiple organ failure and ultimately death. Thus, administration of the correct volume of fluid on a patient basis is critical and may be guided by the proADM value measured for any given patient at any given time during treatment. MR-proADM and lactate behave similarly to the biomarkers in terms of guiding the volume of fluid to be administered during the first 24 hours after diagnosis/therapy initiation. However, MR-proADM is more accurate in guiding the volume of fluid to be administered between 24 and 96 hours.

The method comprises the following steps:

retrospective analysis was performed on 1076 patients with severe sepsis or septic shock from the fluid therapy administered and monitored as detailed above. Later, biomarker values were recorded at baseline (i.e., sepsis diagnosis), 24 hours (day 1), and 96 hours (day 4). Patients were classified as low, moderate or high severity using MR-proADM concentrations at baseline, day 1 and day 4, as described in more detail above:

the total fluid volume administered was then calculated to determine the effect on 28-day mortality and the need for RRT. Within the first 24 hours, blood was collected, then fluid was administered and blood measurements were subsequently made. Similarly, to determine the effect of fluid administration of biomarker kinetics, blood was collected at baseline, fluid was intermittently added for the next 4 days, and then biomarker concentrations were measured again in the blood.

Fluid administration is not performed or adjusted according to the proADM concentration determined during fluid therapy. The evaluation is based on each practitioner treating with the fluid they consider most appropriate without a predetermined layered guideline. Because there is no strict fluid type or volume guideline for sepsis patients, the fluid administered varies significantly across patients, as well as in groups of patients exhibiting similar symptoms.

Effect of fluid administration during the first 24 hours according to MR-proADM kinetics

Patients with consistently lower MR-proADM concentrations receive the lowest fluid volumes. In contrast, a 2-3 fold increase in fluid was administered to patients whose MR-proADM concentration increased from initially moderate to high levels. Similarly, patients with a reduced concentration of MR-proADM (i.e., moderate to low or high to moderate) are administered lower volumes of fluid than those with unchanged severity levels of concentration (i.e., moderate to moderate or high to high).

The results of this analysis are presented in table 36. A moderate correlation (r=0.595) was achieved based on mortality within these MR-proADM severity groups and development of RRT. These results are presented in figure 1.

A strong correlation (r2=0.931) can be found between each MR-proADM severity group, mortality and fluid administration. These results are presented in fig. 2.

Lactate values are also assessed in relation to the amount of fluid administered to the patient. Lactate levels show a similar relationship to fluid volumes compared to proADM. These results are presented in table 37. The correlation was comparable to that of MR-proADM (R ² =0.965), as shown in fig. 3.

Effect of colloidal administration during the first 4 days according to MR-proADM kinetics

A total of 980 patients had MR-proADM values at baseline and day 4. Subsequently, the patients were classified according to their MR-proADM severity as outlined above, and the total volumes of colloidal fluid and blood products were calculated. This is then related to mortality in each group and RRT demand in patients where there is no Renal Replacement Therapy (RRT) at baseline.

The results indicate that increasing fluid administration results in a direct impact on MR-proADM levels based on MR-proADM thresholds, thereby increasing or decreasing the likelihood of mortality from RRT demand. These results are presented in table 38.

A high correlation (r=0.894) was achieved based on RRT requirements and administered fluid volumes within these MR-proADM severity groups, as shown in fig. 4.

Similar results were achieved when the patient's weight was taken into account, resulting in intravenous fluid administration rates in ml/kg and R ² 9.49 as shown in fig. 5.

Lactate values are also assessed in relation to the amount of fluid administered to the patient. Lactate levels show a similar relationship to fluid volumes compared to proADM. These results are presented in table 39. However, the correlation is significantly weaker than (R ² =0.810) MR-proADM, as shown in fig. 6. The accuracy of the correlation between lactate severity group, mortality and total fluid volume administered was significantly lower compared to MR-proADM.

Effect of specific colloid administration during the first 4 days according to MR-proADM kinetics:

administration of individual colloids based on proADM kinetics was assessed from baseline to day 4 in the study population described above.

Gelatin:

a total of 81 (10.6%) patients were administered gelatin during the first 4 days of ICU treatment, with 28-day mortality of 28.4%. The results are presented in table 40.

The results indicate that gelatin appears to be a poor choice for patients with low ADM concentrations. Patients receiving gelatin as a fluid therapy, even with lower levels of proADM, have a higher frequency of mortality than one would expect. Mortality is also high in patients with moderate proADM levels who receive higher gelatin amounts. However, administration of relatively small amounts of gelatin to patients with high proADM levels does result in reduced ADM levels and associated low mortality.

20% albumin:

a total of 144 (18.8%) patients were administered 20% albumin solution during the first 4 days of ICU treatment, with a 28 day mortality of 29.8%. The results are presented in table 41.

The results indicate that 20% albumin appears to be a suitable choice for reducing mortality in patients with high ADM concentrations, but it will also push patients towards RRT needs, as is apparent from the high RRT frequency required for patients receiving relatively large amounts of albumin. In patients with moderate ADM levels administered with relatively small amounts of albumin, mortality and RRT requirements are very low.

HES：

A total of 103 (13.4%) patients were administered a hydroxyethyl starch (HES) solution during the first 4 days of ICU treatment with a 28 day mortality rate of 15.5%. The results are presented in table 42.

A known problem with HES is that administration can lead to increased RRT requirements. This is evident from other studies. The data herein show that this is not the case when the MR-proADM level is low or at a moderate level. When the MR-proADM concentration is already high, RRT is required for all patients administered HES. Thus, HES can be administered (preferably in relatively small amounts) to patients with low or moderate proADM levels at baseline.

Discussion of examples

It is important to evaluate disease severity accurately and rapidly in order to begin the most appropriate treatment as soon as possible. Indeed, delayed or inadequate treatment can lead to widespread worsening of the patient's clinical condition, leading to a greater likelihood that further treatment will become less effective and overall efficacy poorer (8, 27). Thus, a number of biomarkers and clinical severity scores have been proposed to meet this unmet clinical need, currently emphasizing Sequential Organ Failure Assessment (SOFA) scores as the most appropriate tool, allowing it to play a central role in the 2016 sepsis-3 definition (4). This secondary analysis of the SISPCT test (26) compares for the first time sequential measurements of conventional biomarkers and clinical scores such as lactate, procalcitonin (PCT) and SOFA with those of the microcirculation dysfunction marker MR-proADM in a large patient population with severe sepsis and septic shock.

Our results indicate that the first use of MR-proADM within the first 24 hours after sepsis diagnosis yields the strongest correlation with short-, medium-and long-term mortality compared to all other biomarkers or scores. Previous studies largely confirm our findings (17, 28, 29), however conflicting results (30) can be explained in part by the smaller sample volumes analyzed and other factors emphasized in this study (such as microbial species, sources of infection and surgical history before sepsis occurs), all of which affect biomarker performance, thus adding to the latent variability of results in the small study population. Furthermore, our study also closely demonstrates the results of the previous study (17), thus emphasizing the superior performance of MR-proADM in patients with low and medium organ dysfunction severity. Indeed, andaluz-Obeda et al (17) very important view the group of patients with low levels of organ dysfunction, as "this group represents the earliest manifestation in the clinical course of sepsis and/or the lighter form of the disease". Nevertheless, reasonable performance can be maintained across all severity groups with respect to mortality prediction, as is the case across two patient groups defined according to sepsis-3 and septic shock-3 standards.

Analysis of sequential measurements taken after the onset of sepsis allows for the identification of specific patient groups based on disease severity. The identification of both low and high risk patients is of great importance in our analysis. In many ICUs, the demand for ICU beds can periodically exceed availability, which can lead to insufficient triage, resource dosing, and a subsequent decrease in the likelihood of proper ICU occupancy (32-35). Thus, accurate assessment of patients with low risk of hospitalization mortality who may be eligible to pass from the ICU to the general ward would be significantly beneficial. At each time point measured in our study, MR-proADM can identify the ICU, hospitalization and higher number of low severity patients with the lowest 28-day mortality. Further analysis of the patient group with low severity and no additional ICU-specific therapies indicated that an additional 4 day ICU hospitalization was observed at each time point after biomarker measurement. The biomarker driven method for accurately identifying low severity patients reduced 28-day mortality and 90-day mortality compared to the patient population that was actually admitted to leave at each time point. Indeed, patients who are admitted to leave have various low, medium and high severity MR-proADM concentrations, which are subsequently reflected in higher mortality rates. However, it is not clear whether many patients in this group still need further ICU treatment for non-microcirculation, non-life threatening problems, or whether a bed is available in an ordinary ward. Nonetheless, such biomarker driven grant-away ICU methods plus clinician judgment can improve correct stratification of patients, with concomitant clinical benefit and potential cost savings.

Conversely, identification of high risk patients who may require early and targeted therapy to prevent subsequent clinical deterioration may have even greater clinical relevance. Considerable cost savings and reduction in antibiotic use have been observed with PCT-guided algorithms in SISPCT studies and other trials (26, 36, 37), however, relatively high mortality rates may be observed even when PCT values appear to steadily decrease. Our study showed that adding MR-proADM to a model of PCT decreasing over the following ICU days allowed for the identification of low, medium and high risk patient groups, wherein increasing and decreasing MR-proADM severity levels from baseline to day 1 provided a sensitive early indication of treatment success. In addition, predicting the need for future focal clearance or emergency surgery and susceptibility to the occurrence of new infections would be of substantial benefit in initiating additional therapeutic and interventional strategies in an attempt to prevent any future clinical complications at an early stage.

The advantages of our study include a comprehensive examination of several different subgroups of low and high disease severity from the randomized trial database, thereby adjusting for potential confounding factors and including maximum sample size of sepsis (characterized by definition of sepsis 1 and sepsis 3) patients and information on MR-proADM kinetics. In summary, MR-proADM is superior to other biomarkers and clinical severity scores both at the time of initial diagnosis and during ICU treatment in terms of its ability to identify mortality risk in sepsis patients. Thus, MR-proADM may be used as a tool to identify high severity patients who may require alternative diagnostic and therapeutic interventions and combining low severity patients for whom the absence of ICU-specific therapies may potentially qualify to be admitted off the ICU in advance. The analysis presented herein also enables therapy guidance for fluid therapy based on proADM measurements.

Watch (watch)

TABLE 1 patient characteristics at baseline for survival up to 28 days

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ICU: an intensive care unit; COPD: chronic obstructive pulmonary disease; MR-proADM, a middle adrenomedullin precursor; PCT: procalcitonin; CRP: c, reacting protein; SOFA: sequential organ failure assessment; SAPS II: simplifying acute physiology scoring; APACHE II: acute physiology and chronic health assessment. The data are presented in absolute values and percentages in brackets, indicating the proportion of surviving patients and non-surviving patients at 28 days.

TABLE 2 prediction of mortality 28 days after sepsis diagnosis

N: number of pieces; AUROC: area under the subject's working curve; LR χ2: HR: hazard ratio; IQR: a quarter bit difference. All multivariate analyses were correlated to 28-day mortality by p < 0.0001.

TABLE 3 survival analysis for 7 day mortality, 90 day mortality, ICU mortality, and hospitalization mortality

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Regarding 7-day mortality, all multivariate p values < 0.0001 except PCT and CRP (0.0015 and 0.0843, respectively).

TABLE 4 survival analysis of MR-proADM when added to individual biomarkers or clinical scores

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HR IQR [95% CI ] indicates the hazard ratio of MR-proADM in each bivariate or multivariate model. The degree of freedom of each bivariate model is 2, in contrast to 11.

TABLE 5 AUROC analysis of 28 day mortality prediction based on SOFA severity level

N: number of pieces; AUROC: area under the subject's working curve; LR χ2: HR: hazard ratio; IQR: a quarter bit difference.

TABLE 6 survival analysis of MR-proADM in different organ dysfunction severity groups in combination with biomarkers alone or clinical scores

TABLE 7 severity of disease group for corresponding 28-day SOFA and MR-proADM

MR-proADM: middle adrenomedullin precursor; SOFA: sequential organ failure assessment

TABLE 8 corresponding 28 day SAPS II and MR-proADM disease severity groups

MR-proADM: middle adrenomedullin precursor; SAPS II: simplifying acute physiology II

TABLE 9 corresponding 28 day APACHE II and MR-proADM disease severity groups

MR-proADM: middle adrenomedullin precursor; APACHE II: acute physiological and chronic health assessment II

TABLE 10 severity of disease group for corresponding 28-day lactate and MR-proADM

MR-proADM: middle adrenomedullin precursors

TABLE 11 correlation of biomarkers and SOFA with 28-day mortality on days 1, 4, 7 and 10

TABLE 12 Low and high risk severity groups throughout ICU treatment and corresponding mortality rates

TABLE 13 mortality and duration of ICU therapy based on MR-proADM concentration and ICU specific therapy

* Mortality not involving the same day or the next day

TABLE 14 time-dependent Cox regression of single and cumulative additions of MR-proADM

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MR-proADM: middle adrenomedullin precursor; DF: degree of freedom

TABLE 15 28 day mortality and 90 day mortality according to PCT and MR-proADM kinetics

The hazard ratio for patients with the following: * Lasting medium value versus low value; * Sustained high value contrast medium value; * Sustained high vs low;low value increases to medium value contrast continuously low value; />The medium value is increased to the high value and the continuous medium value is compared; />The high value decreases to a medium value versus a continuously high value; />The medium value is reduced to a low value and the medium value is increased to a high value. Kaplan Meier plots show individual patient subgroups or individual patient subgroups grouped into ascending or descending subgroups.

TABLE 16 mortality after PCT concentration and MR-proADM severity level changes

The hazard ratio for patients with the following: * Lasting medium value versus low value; * Sustained high value contrast medium value; * Sustained high vs low;low value increases to medium value contrast continuously low value; />Medium value increases to high value pairConstant value of the ratio; />The high value decreases to a medium value versus a continuously high value; />The medium value is reduced to the low value and the continuous medium value

TABLE 17 28 day mortality and 90 day mortality after PCT concentration and MR-proADM severity level changes

The hazard ratio for patients with the following: * Lasting medium value versus low value; * Sustained high value contrast medium value; * Sustained high vs low;low value increases to medium value contrast continuously low value; />The medium value is increased to the high value and the continuous medium value is compared; />The high value decreases to a medium value versus a continuously high value; />The medium value is reduced to the low value and the continuous medium value

TABLE 18 ICU mortality and hospitalization mortality after changes in PCT concentration and MR-proADM severity levels

The hazard ratio for patients with the following: * Lasting medium value versus low value; * Sustained high value contrast medium value; * Sustained high value contrastA low value;low value increases to medium value contrast continuously low value; />The medium value is increased to the high value and the continuous medium value is compared; />The high value decreases to a medium value versus a continuously high value; />The medium value is reduced to the low value and the continuous medium value

TABLE 19 influence of infectious sources on the 28 day mortality prediction

In pulmonary sources of infection, the MR-proADM AUROC value is significantly greater than all other parameters except APACHE II.

TABLE 20 influence of microorganism species on the 28 day mortality prediction

TABLE 21 influence of modes of entering ICU on 28 day mortality prediction

TABLE 22 correlation of biomarkers and clinical scores with SOFA at baseline and day 1

* Patients who used the same patients on baseline as on day 1

TABLE 23 correlation of baseline MR-proADM with SOFA sub-scores at baseline and day 1

TABLE 24 correlation of biomarkers with SOFA score throughout ICU treatment

TABLE 25 mortality-baseline to day 1 based on MR-proADM severity and elevated or reduced PCT concentrations

Table 26 PCT kinetics from baseline to day 1-new infections occurred on days 1, 2, 3, 4.

Table 27 PCT kinetics from baseline to day 4-new infections occurred on days 4, 5, 6, 7.

Table 28 PCT kinetics from baseline to day 1-lesion clearance requirements on days 1, 2, 3, 4.

Table 29 PCT kinetics from baseline to day 4-lesion clearance requirements on days 4, 5, 6, 7.

Table 30 PCT kinetics from baseline to day 1-emergency surgical requirements on days 1, 2, 3, 4.

TABLE 31 antibiotic changes from baseline to day 1 elevated PCT-day 4

TABLE 32 antibiotic changes from baseline to day 4 elevated PCT-day 4

TABLE 33 biomarker levels based on platelet count

TABLE 34 platelet count based on proADM levels

TABLE 35 thrombocytopenia occurrence and proADM kinetics at baseline and day 1

Table 36.

Table 37.

Table 38.

Table 39.

Baseline ADM	ADM day 4	N	Number of deaths	Weight-adjusted fluid volume
					Low and low	Low and low	45	13.3％	10.60ml/kg
Low and low	Medium and medium	108	9.3％	11.51ml/kg
					Medium and medium	Medium and medium	376	17.3％	13.45ml/kg
Medium and medium	Low and low	74	9.5％	12.42ml/kg
					Medium and medium	High height	92	43.5％	22.28ml/kg
High height	High height	35	51.4％	42.27ml/kg
					High height	Medium and medium	59	18.6％	19.42ml/kg

Table 40.

Table 41.

Table 42.

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Claims

1. Use of a detection reagent binding to adrenomedullin precursor (proADM) or a fragment thereof for the preparation of a product for a method of therapy guidance, stratification and/or monitoring of fluid therapy in a patient having been diagnosed with sepsis, severe sepsis or septic shock, the method comprising

Providing a sample of the patient in question,

determining the level of proADM or a fragment thereof in said sample, wherein said fragment thereof is MR-proADM,

wherein the level of proADM or a fragment thereof indicates a prescription of fluid therapy to be administered to the patient, wherein the prescription of fluid therapy comprises a prescription of volume, frequency and/or rate of therapeutic fluid to be administered to the patient,

wherein the patient receives fluid therapy and a medium or high severity level of proADM or a fragment thereof measured in the sample indicates modification or adjustment of the fluid therapy, including reducing the volume and/or rate of the fluid to be administered to the patient, adjusting the fluid therapy to minimize risk or improve the health of the patient or prevent adverse events from occurring,

Wherein the moderate severity level of proADM or a fragment thereof is from 2.75.+ -. 20% nmol/l to 10.9.+ -. 20% nmol/l,

and wherein the high severity level of proADM or a fragment thereof is higher than 10.9 ± 20% nmol/l.

2. The use of claim 1, wherein the patient has been diagnosed with one or more organ failure, and/or wherein the patient is a post-traumatic or post-operative patient.

3. The use of claim 1, wherein the fluid therapy comprises administration of a colloidal solution, or blood, or a blood-derived fluid.

4. The use according to claim 3, wherein the colloidal solution is selected from the group consisting of gelatin, albumin and starch solutions.

5. The use of claim 1, wherein the fluid therapy comprises administration of a crystalline solution.

6. The use of claim 1, further comprising determining lactate levels in a sample isolated from the patient.

7. The use according to claim 1, wherein the patient is receiving fluid therapy and a medium or high severity level of proADM or a fragment thereof determined in the sample is indicative of an adverse event,

8. The use according to claim 1, comprising

Determining the level of proADM or a fragment thereof in a first sample and a second sample from the patient, wherein the second sample is obtained after obtaining the first sample;

wherein no change or an increase in the level of proADM or a fragment thereof in the second sample compared to the first sample is indicative of a decrease in the volume, frequency and/or rate of the fluid to be administered to the patient,

or alternatively

Wherein an increase in proADM or a fragment thereof from a low to a medium or high severity level or an increase in proADM or a fragment thereof from a medium to a high severity level is indicative of a decrease in the volume and/or rate of the fluid to be administered to the patient, wherein

The low severity level of proADM or fragments thereof is below 2.75.+ -. 20% nmol/l,

the moderate severity level of proADM or fragments thereof is from 2.75.+ -. 20% nmol/l to 10.9.+ -. 20% nmol/l, and

the high severity level of proADM or fragments thereof is higher than 10.9.+ -. 20% nmol/l.

9. The use of claim 8, wherein the first sample is isolated at or before the onset of fluid therapy, i.e., time point 0, and the second sample is isolated at a time point between 12 hours and 36 hours after the onset of therapy, or at a time point between day 3 and 5 days after the onset of therapy.

10. The use of claim 1, wherein the patient receives fluid therapy and

a low severity level of proADM or a fragment thereof determined in the sample indicates that 2.78 + 20% ml/kg or less of fluid is administered to the patient within 24 hours, wherein ml/kg represents the volume of fluid in ml administered per kilogram of body weight of the patient,

a moderate severity level of proADM or a fragment thereof measured in the sample indicates that 4.94 ± 20% ml/kg or less of fluid is administered to the patient within 24 hours, or

A high severity level of proADM or a fragment thereof measured in the sample indicates that 9.95 ± 20% ml/kg or less of fluid is administered to the patient within 24 hours,

and is also provided with

Wherein the low severity level of proADM or fragments thereof is below 2.75.+ -. 20% nmol/l,

11. Use of a pharmaceutical composition comprising a therapeutic fluid for the preparation of a medicament in fluid therapy of a patient in need thereof, wherein administration of said composition to said patient is performed after prescribing said administration according to the method of claim 1.

12. The use of claim 11, wherein the pharmaceutical composition comprises a colloidal or crystalline solution.

13. The use according to claim 12, wherein

The patient has received fluid therapy and

decreasing the volume, frequency and/or rate of the fluid administered to the patient that the patient receives when the proADM or fragment thereof in the patient's sample is determined to be of medium or high severity level,

and wherein the high severity level is above 10.9 + -20% nmol/l,

or alternatively

Wherein the level of proADM or a fragment thereof in a first sample and a second sample from the patient has been determined, wherein the second sample is obtained after obtaining the first sample;

wherein the volume, frequency and/or rate of the fluid administered to the patient is reduced when the level of proADM or a fragment thereof in the second sample is determined to be unchanged or increased from the first sample.

14. Use according to claim 12 or 13, wherein

Administering 2.78+ -20% ml/kg or less of fluid to a patient of low severity level in 24 hours of proADM or a fragment thereof in said sample, wherein ml/kg represents the volume of fluid in ml administered per kilogram of body weight of said patient,

Administering 4.94+ -20% ml/kg or less of fluid to a patient of moderate severity level in 24 hours of proADM or a fragment thereof in said sample,

administering 9.95.+ -. 20% ml/kg or less of fluid to a patient whose proADM or fragment thereof in said sample is determined to be of high severity level within 24 hours,

and is also provided with

15. The use according to claim 12 or 13, wherein a starch solution is administered to the patient when the low and/or medium severity level of proADM or a fragment thereof determined in the sample is determined.

16. The use according to claim 12 or 13, wherein when the high severity level of proADM or a fragment thereof determined in the sample is determined, an albumin solution is administered to the patient.

17. The use according to claim 12 or 13, wherein a gelatin solution is administered to the patient when the medium or high severity level of proADM or a fragment thereof determined in the sample is determined.

18. The use of claim 12 or 13, wherein the patient receives intravenous administration of the composition.

19. Kit for use according to claim 1, comprising:

a detection reagent for determining the level of proADM or a fragment thereof in a sample from a subject, wherein the fragment thereof is MR-proADM,

reference data corresponding to a high, medium and/or low severity level of proADM, comprising a reference level, wherein the low severity level of proADM or a fragment thereof is below 2.75 ± 20% nmol/l, wherein

A moderate severity level of proADM or a fragment thereof of 2.75 ± 20% nmol/l to 10.9 ± 20% nmol/l, and wherein a high severity level of proADM or a fragment thereof is above 10.9 ± 20% nmol/l, wherein the reference data is stored on a computer readable medium and/or used in the form of computer executable code configured to compare the determined levels of proADM or a fragment thereof, and

a pharmaceutical composition comprising a therapeutic fluid.

20. The kit of claim 19, wherein the pharmaceutical composition comprises a colloid.

21. The kit of claim 19, wherein the pharmaceutical composition comprises a crystalline solution.